The film opens at the Belle Reve Penitentiary. Floyd Lawton (AKA: Deadshot) (Will Smith) is in his cell going at his punching bag. The guards, led by Captain Griggs (Ike Barinholtz), bring Floyd a lousy meal. When Griggs taunts him, Floyd threatens to get him someday. Taking that as a threat, Griggs has Floyd strapped to a chair and beaten by the other guards.
Meanwhile, Harley Quinn (Margot Robbie) is hanging upside down in her cell (more like a cage). Griggs shows up to see her, and he has one of the guards turn on the electricity on the bars when she gets too close. This triggers a flashback in Harley's mind of when she first showed up at the place, having just gone insane.
It's been months since the death of Superman (a reference to the film 'Batman vs. Superman'). Amanda Waller (Viola Davis) meets with her associates Admiral Olsen (Ted Whittall) and Dexter Tolliver (David Harbour) to discuss the organization of Task Force X, her idea of creating a team of the most dangerous criminals to combat any possible major catastrophe. Waller pulls out her files and goes through her proposed recruits.
She starts with Floyd, who was an expert marksman known as Deadshot. In a flashback, we see Deadshot preparing to take out a target. He goads his employer into doubling his pay, and Deadshot kills his target by having his bullet ricochet off a gate to strike the man. Floyd has a daughter, Zoe (Shailyn Pierre-Dixon). In another flashback, the two were walking together and discussing Zoe moving in with Floyd. Her mother wouldn't let her because they both know Floyd is a killer, though he denied it to Zoe. He was caught by Batman (Ben Affleck). Floyd got his gun and prepared to shoot Batman, but Zoe stood in the way and pleaded with him not to do it. Floyd relented and went quietly.
Waller's next pick is Harley, who was originally a therapist named Dr. Harleen Quinzel. Her last patient was The Joker (Jared Leto). Instead of fixing him, she fell in love with him. The Joker escaped from prison by using Harleen as a hostage. He then tortured Harleen for months until she went insane and turned her into his love slave and eventually his wife. Waller knows that Harley is even crazier and more fearless than Joker, which makes her a very valuable asset. Another couple flashbacks show Joker and Harley in a nightclub where Harley was dancing. He invited her to join him and a gangster he met with named Monster T (Common). Harley offered him company, but he declined out of fear that Joker would be displeased. This got him killed. Later, Joker and Harley made their getaway but were chased by Batman in the Batmobile. The two were ran off the road and into the river. Batman jumped in and found Harley, then took her to jail.
Among Waller's other picks are Digger Harkness (AKA: Captain Boomerang) (Jai Courtney), a thief that used a bladed boomerang and would double-cross his partners. He was caught by The Flash (Ezra Miller). Next is Chato Santana (Jay Hernandez), a former gang-banger known as El Diablo. He possesses pyrokinetic abilities, and he willingly turned himself in. Then there's Waylon Jones/Killer Croc (Adewale Akinnouye-Agbaje), a thug with an unusual disorder that gives him green scaly skin. Croc was treated like a monster, so he acted like one and devoured anyone that crossed him. Waller's last pick is Dr. June Moone (Cara Delevignge), an archaeologist who recently came across a cave and found an idol, releasing the spirit of a 6,000-year-old witch called Enchantress. The spirit possesses June, but Waller carries the heart of Enchantress with her in order to maintain control over her. Helping Waller is Colonel Rick Flag (Joel Kinnaman), who is motivated because he is in love with June.
Waller, Tolliver, and others gather at the Pentagon in Washington DC to discuss the initiation of Task Force X. The chairman is not feeling the plan until Waller tells June to bring out Enchantress. She morphs into the witch and demonstrates her power, with Waller poking the heart to control her, which pains Enchantress and also hurts June once she returns to normal. Waller tells the others in the room that the plan would be for her team to go out in secrecy with no word of the government's involvement, and if the villains get caught, they'll get "thrown under the bus".
Waller heads to Belle Reve with Flag and June to continue recruiting. She brings Floyd out and allows him to use an array of guns for target practice. He gives Flag a list of demands, including freedom from his sentence and custody of his daughter. Flag refuses to give into these demands, but Floyd says he was talking to Waller.
At night, June morphs into Enchantress. Flag is trying to call Amanda, but Enchantress hits Flag's mind with a vision of June dead to terrify him. Enchantress then teleports to Waller's home and finds an idol that contains the spirit of her brother, Incubus. She takes the idol and heads to the Midway City train station and finds a commuter (Alain Chanoine) and uses his body to bring Incubus back to life. Reunited, the two discuss Enchantress's plan to create a machine to wipe out humanity. Incubus, still in the commuter's body, kills three men on the tracks before taking his true form and causing destruction.
Waller is alerted to the chaos and decides it's time to activate Task Force X. The villains are taken from their cells and injected with nano bombs that will go off if they fail or try to escape. They are brought to a base where they are briefed on their mission, which is to head into Midway City and retrieve a mark. The last recruit, Slipknot (Adam Beach), arrives, where he first punches out an FBI agent. When Flag tells the villains about their set-up, Floyd refers to them as a "Suicide Squad". They are given proper outfits and their own weapons to head into battle. Lastly, Flag shows them a tablet with Waller speaking to them and telling them what the deal is. On the ride there, the squad is joined by Flag's bodyguard, Tatsu Yamashiro (AKA: Katana) (Karen Fukuhara), a lethal Japanese assassin whose sword contains the souls of those that she kills. Her husband was killed with that sword and his own soul is trapped in there.
Meanwhile, one of Joker's henchmen finds him in his apartment surrounded by his weapons to tell him about Harley's situation. Joker goes to find Griggs in a casino and tortures him until he tells him everything. Joker and his goons then attack the Van Criss research facility where the nano bombs were produced. Joker shows the head doctor that he's kidnapped his wife to blackmail him into helping him.
Incubus turns Enchantress into a more powerful form when Waller repeatedly stabs her heart. As the squad is flown into Midway City, Enchantress' soldiers shoot them down and causes their chopper to crash-land. They walk to their destination, but Slipknot attempts to make an escape. He shoots his grappling hook to get out, but Flag sets off his nano bomb and kills Slipknot, proving he was not bluffing.
The squad is attacked by mutated creatures (formerly human males) under the bidding of Enchantress, prompting them to utilize their capabilities. Deadshot hits every target, Harley shoots and whacks the minions with her baseball bat, Katana slices them with her sword, and Boomerang uses his weapon, but Diablo sits this one out. Flag is grabbed by the minions, and Harley almost lets him die, but Deadshot reminds her that if he dies, then they all die. She reluctantly saves Flag and kills the minions.
Flag leads the squad to a building to find their mark. Harley takes an elevator and is attacked by more minions. She easily kills them before rejoining the squad. More minions attack them, so Deadshot pushes Diablo into unleashing the full might of his fire powers. Diablo incinerates all the minions with one blast.
As they walk to their destination, Harley looks down the staircase and she remembers the night she became what she is now. Joker brought her to a chemical plant and convinced her to "live" for him. She willingly tossed herself into the vat of chemicals, and Joker jumped in after her, pulling her out and laughing maniacally.
In the present, the squad finds a room that Flag enters to find Waller waiting for him. As they discuss their mission in front of four other agents, Waller kills them all since they know too much. The rest of the squad is surprised to find Waller there. Together, they all go to the rooftop for extraction. Unfortunately, the chopper they're waiting for has been hijacked by Joker and his men. One of the men deactivates the nano bomb in Harley's neck, allowing her to escape with Joker. Waller orders Deadshot to shoot her down with the promise of custody of his daughter. Deadshot shoots but intentionally misses Harley. Waller then orders her men to shoot the chopper down. Harley falls out of the chopper and lands on a rooftop, and she watches as Joker goes down with the chopper.
Waller is taken by Enchantress's minions and brought to her, allowing Enchantress to get inside Waller's mind and learn what she already knows. Enchantress also gets her heart back, giving her more power.
Harley rejoins the squad when they come across her on their path. Deadshot finds Waller's case file and learns the truth about their mission. He demands an explanation from Flag. Flag admits that three days earlier, he and June went underground together to set something up, but she morphed into Enchantress and abandoned him. The squad ditches Flag as Deadshot heads into a bar to get a drink.
In the bar, the squad drinks together with Harley acting as the barmaid in serving them drinks. Deadshot tells them that he knows they're just pawns in Waller's plan and that they are all expendable. During the conversation, Diablo discusses why he stopped using his powers. He used to have a family, but his wife Grace (Corina Calderon) found out about his life as a gangster, and in a fit of rage, Diablo burned their house down with Grace and their two kids inside. Flag then enters the bar to tell the squad they can go free if they choose, as he breaks the detonator to their bombs. He gives Deadshot letters from his daughter, showing she had written to him everyday. This gives Deadshot the motivation to continue the mission, leading the rest of the squad to join him.
The plan is to plant a bomb beneath the station to take out Incubus so that the rest of the squad can get close enough to Enchantress. Croc joins Flag's men to head into the sewers and place the bomb. The squad hides, but Enchantress senses them and offers them the things they want the most as she goes into their minds. She shows Deadshot a vision of him killing Batman. Harley sees her and Joker looking normal and starting a family. Flag sees himself with June. Diablo sees himself with his family again. Only he realizes quickly that it's a trick. He heads into the fight against Incubus, hurling his fire powers at him before morphing into his true form, a towering fire creature as big as Incubus. Diablo roasts Incubus and holds him in the corner long enough for Croc and the others to place the bomb. Diablo stays there to hold Incubus down as the bomb explodes, killing both of them.
The now enraged Enchantress takes on the whole squad, with Croc returning to join the fight. But the witch is far too powerful and evasive for any one of them to get her, so she offers them what they want if they join her. Harley appears to go through with it and approaches Enchantress. However, Harley tricks Enchantress and uses a sword to cut her heart out. Croc then throws a bag of explosive in the path of Enchantress's machine, as Deadshot gets ready to shoot at it. Enchantress tries to trick him by making it look like Zoe is there to beg him to not shoot, but Deadshot pulls the trigger and hits the bombs, destroying the machine. Enchantress is now weakened, and Flag holds her heart. He orders the witch to release June, but Enchantress says she won't come back. Flag crushes the heart and kills Enchantress. June peels off the witch's skin, returning to normal and embracing Flag. As the squad prepares to leave, Waller emerges with her own detonator. She says they only get 10 years off their sentences, with some special privileges.
Floyd is allowed to visit Zoe for a brief while before being escorted out by Flag. Croc is allowed to have cable TV in his cell. Boomerang gets moved into solitary confinement for his demands. Harley is given as espresso machine in her cell. Moments later, a group of mercs breaks into the penitentiary and start killing the guards before breaking into Harley's cell. The Joker (STILL ALIVE) enters and embraces Harley, whispering into her ear, "Let's go home..."
During the closing credits, Waller meets with Bruce Wayne in a restaurant. She asks him for protection in the aftermath of Enchantress's destruction, and in exchange, she hands him case files on Barry Allen (Flash) and Arthur Curry (Aquaman). As Bruce leaves, Waller comments that he should "stop working nights". Bruce says she should shut her operation down, since he and his new friends will be taking care of things.