According to the interview on Shout! Factory's first DVD box set, Lou Albano had stated that he was not interested in portraying Mario and voicing him at first, but was talked into it by his wife.
Princess Peach Toadstool:
More fasty, more fasty, Uncle Luigi.
King Bowser Koopa: Stop or I'll tell on you. I've been hanging around these kids too long.
King Bowser Koopa: Net *them*, you idiots! Not me!
In the opening credits when Mario and Luigi run across the screen, Luigi noticeably slows down as he reaches his "mark" at the end.
Lou Albano, in live-action, sings and dances to "Do The Mario" during the closing credits.
When shown in reruns after cancellation, DiC took out all the song covers played during the action/chase sequences, and replaced them with instrumentals of songs featured in The Adventures of Super Mario Bros. 3 (1990) and Super Mario World (1991), presumably because of music licensing issues for each of the songs. The original songs have been reinstated in the UK DVD volumes, but not on the Region 1 DVD releases, not even the box sets.