- Dr. John Dolittle as Mario
- Grandpa Archer Dolittle as Yoshi
- Dr. Mark Weller as Luigi
- Calloway as Toad
- Lucky the Dog as Oogtar
- Rodney the Guinea Pig as Detective Herlock Solmes
- Dr. Gene "Geno" Reiss as Wario
- Lisa Dolittle as Princess Daisy
- Dr. Fish as Waluigi
- Rat #1 as Bowser
- Rat #2 as Donkey Kong
- Old Beagle as Bowser Jr.
- Baby Tiger as Ludwig Von Koopa
- Maya Dolittle as Princess Peach
- Charisse Dolittle as Princess Rosalina
- Possum as Diddy Kong
- Bettleheim the Cat as Morton Koopa, Jr.
- Male Pigeon as Boo
- and more
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