Peter Parker / Spider-Man (26 episodes, 2008-2009)
Gwen Stacy (25 episodes, 2008-2009)
Flash Thompson / Car Thief #1 / Nick Bottom (21 episodes, 2008-2009)
Sally Avril / Betty Brant / Voice of OsCorp / Coffee Clerk / Computer Voice / Erin / Stephanie Briggs (20 episodes, 2008-2009)
Liz Allan / Helena / Trina (19 episodes, 2008-2009)
Mary Jane Watson / Hermia / Rosie Thompson (18 episodes, 2008-2009)
Harry Osborn / Frederick Foswell / Patch / Alan O'Neil / Homunculi (17 episodes, 2008-2009)
George Stacy / Rhino / Alex O'Hirn / Ox (16 episodes, 2008-2009)
May Parker / Aunt May Parker (16 episodes, 2008-2009)
Green Goblin / Dilbert Trilby / Seymour O'Reilly / Blackie Gaxton / Chameleon / Homunculi / Dilbert Trillby / Homunculus #3 / Matthew Besnick / Mr. Parrot (15 episodes, 2008-2009)
Norman Osborn (15 episodes, 2008-2009)
Kenny Kong / Ned Lee / Bodyguard / Lab Assistant / Oberon (14 episodes, 2008-2009)
Rand Robertson / Robbie Robertson / Fancy Dan / Homunculus #1 / Randy Robertson / Ranger / Ricochet / Ship Helmsman (14 episodes, 2008-2009)
Eddie Brock / Venom (13 episodes, 2008-2009)
J. Jonah Jameson / John Jameson / Colonel Jupiter (13 episodes, 2008-2009)
Hammerhead / Sandman / Flint Marko / Car Thief #2 (12 episodes, 2008-2009)
Dr. Otto Octavius / Master Planner / Doctor Octopus / Dr. Octopus / Otto Octavius (12 episodes, 2008-2009)
Tombstone / Principal Davis / Big Man / Coach Smith / L. Thompson Lincoln / OsCorp Security Guard / Ship Captain / Thug (12 episodes, 2008-2009)
Curt Connors / Dr. Curt Connors / Homunculi / Lizard (9 episodes, 2008-2009)
St. John Devereaux / Montana / Shocker / Bernard Houseman (9 episodes, 2008-2009)
Martha Connors / Anna Watson / Trina (9 episodes, 2008-2009)
Miles Warren / Aaron Warren (7 episodes, 2008-2009)
Electro / Clerk / Max Dillon / Thug #1 (6 episodes, 2008-2009)
Glory Grant / Cobweb / Homunculus #2 (6 episodes, 2008-2009)
Stan Carter (6 episodes, 2008-2009)
Sha Shan Nguyen / Titania (5 episodes, 2009)
Mark Allan / Molten Man (5 episodes, 2009)
Tinkerer / Homunculi / Phineas Mason (4 episodes, 2009)
Jean DeWolff (4 episodes, 2008-2009)
Quentin Beck / Mysterio (4 episodes, 2008-2009)
Doc Bromwell (4 episodes, 2008-2009)
Vulture / Adrian Toomes (4 episodes, 2008-2009)
Donald Menken (4 episodes, 2009)
Black Cat / Felicia Hardy (3 episodes, 2008-2009)
Silver Sable / Sable Manfredi (2 episodes, 2009)
Silvermane / Silvio Manfredi (2 episodes, 2009)
Ashley Kafka (2 episodes, 2008-2009)
Kraven / Kraven the Hunter / Sergei Kravinoff (2 episodes, 2009)
Ben Parker (1 episode, 2008)
Billy Connors (1 episode, 2008)
Sullivan Edwards (1 episode, 2008)
Calypso Enzili (1 episode, 2009)
Crusher Hogan (1 episode, 2008)
Big Man (1 episode, 2008)
Car Salesman / Ted Twaki (1 episode, 2008)
Morris Bench (1 episode, 2009)
Hobie Brown / Puck (1 episode, 2009)
Stan (1 episode, 2009)
Mayor Waters (1 episode, 2008)
Joan Jameson (1 episode, 2009)
Kingsley (1 episode, 2009)
Ox (1 episode, 2009)
Cat Burglar / Walter Hardy (1 episode, 2009)
Dude with Cell Phone (uncredited) (1 episode, 2008)