Bart: Bart is one of the playable characters, he can jump, kick, use his slingshot, open his cape to release bats and turn into Bartman to glide, climb walls and ziplines, grapple and (only in the Xbox version) turn into Robo-Bart.
Lisa: Lisa is one of the playable characters, she can jump, kick, play her saxophone to stun enemies, use Buddha's Hand to pick up and drop big things and kick enemies and (only in the Xbox version) turn into Clobber Girl.
Professor Frink: he is kidnapped and placed on a platform by Donkey Kong until Bart and Lisa save him.
Donkey Kong: he kidnaps Professor Frink and escapes through the warp pipes. He is the final boss of the level and Lisa defeats him by kicking him to the goo.
Koopa Troopa: she is Frink's new girlfriend and, by him, she's very funny.
Karate masters: enemies throughout the level, who can kick and throw fire balls at you.
Footbal players: more enemies, who can run and push you and throw football balls at you.
Mario: a video game character who can be seen in the factory working sadly.
Sonic the Hedgehog: another video game character seen working sadly in the factory, later thrown to the goo by Bart.