- Mario as Thomas
- Luigi as Edward
- Chunky Kong as Henry
- Donkey Kong as Gordon
- Oogtar as James
- Yoshi as Percy
- Toad as Toby
- Diddy Kong as Duck
- Mallow as Donald
- Geno as Douglas
- Green Toad as Oliver
- Bowser as Diesel
- Koopa Troopa as Bill
- Koopa Paratroopa as Ben
- Link (from The Legend of Zelda) as BoCo
- Wendy O. Koopa as Daisy
- Birdo as Mavis
- Yellow Toad as Stepney
- Dixie Kong as Emily
- Shy Guy as Bertie
- Funky Kong as Salty
- Banjo (from Banjo) as Harvey
- Wario as Arry
- Waluigi as Bert
- Blue Toad as Fergus
- Baby Mario as Skarloey
- Baby Luigi as Rheneas
- Donkey Kong Jr. as Sir Handel
- Baby Yoshi as Peter Sam
- Kirby (from Kirby) as Rusty
- Bowser Jr. Duncan
- Fox McCloud (from Star Fox) as Duke
- General White as Bertram
- Goldbob as Proteus
- Professor Frankly as Freddie
- Blue Yoshi & Shy Blue Yoshi as Mighty Mac
- Red Toad as Arthur
- Zelda (from The Legend of Zelda) as Lady
- Smithy as Diesel 10
- Morton Koopa Jr. as Splatter
- Larry Koopa as Dodge
- Cranky Kong as Sir Topham Hatt
- Wrinkly Kong as Lady Hatt
- Orange Yoshi as Terence
- Toadbert as Trevor
- White Yoshi as Toad
- R.O.B. as Derek
- Iggy Koopa as Bulgy
- Candy Kong as Elizabeth
- Boshi as George
- Detective Herlock Solmes as Murdoch
- King Boo as Spencer
- Tiny Kong as Caroline
- Big Mickey (from TUGS) as Cranky
- Boo as Scruffey
- Professor E. Gadd as Mr. Percival
- A White Shy Guy as Harold
- Princess Peach as Rosie
- The Goombas as The Troublesome Trucks
- Baby Bowser as Smudger
- Robo Koopa as Bulstrode
- Princess Rosalina as Molly
- Baby Peach & Baby Daisy as Annie & Clarabel
- Sonic (from Sonic The Hedgehog) as Jack
- Tails (from Sonic The Hedgehog) as Alfie
- Uncle Chuck (from Sonic The Hedgehog) as Nelson
- Lucas (from Sonic The Hedgehog) as Oliver (Pack)
- Scratch & Grounder (from Sonic The Hedgehog) as Max & Monty
- Chuck Thorndyke (from Sonic The Hedgehog) as Kelly
- Knuckles (from Sonic The Hedgehog) as Byron
- Esipo (from Sonic The Hedgehog) as Ned
- Amy Rose (from Sonic The Hedgehog) as Isobella
- Antoine (from Sonic The Hedgehog) as Patrick
- Rotor (from Sonic The Hedgehog) as Buster
- Queen Acorn (from Sonic The Hedgehog) as Miss Jenny
- King Acorn (from Sonic The Hedgehog) as The Foreman
- Olimar (from Olimar) as Whiff
- Bob-omb Buddy as Scruff
- Luma as Dennis
- Mighty Moe (from TUGS) as Rocky
- Purple Toad as Charlie
- Black Yoshi as Neville
- Princess Daisy as Belle
- Fawful as D261
- Red Koopa Kid as Lorry 1
- Green Koopa Kid as Lorry 2
- Blue Koopa Kid as Lorry 3
- Falco (from Star Fox) as Victor
- Slippy (from Star Fox) as Kevin
- Bottles (from Banjo) as Bash
- JamJars (from Banjo) as Dash
- Mumbo Jumbo (from Banjo) as Ferdinand
- Alley Koop as The Spiteful Breakvan
- Madame Flurrie as Madge
- Lanky Kong as Hank
- Toadette as Flora
- Queen Bee as Old Slow Coach
- Ms. Mowz as Henrietta
- Kiddy Kong as Billy
- Rudy the Clown as Hector
- Toadsworth as Hiro
- Henry as Den
- Orville as Dart
- Mr. Game & Watch as Captain
- Squirtle (from Pokemon) as Flynn
Special Guests from Big Machine Records
- Peter as Mr. Conductor (Alec Baldwin)
- Brian as Mr. Conductor Junior
- Herbert as Burnett Stone
- Meg as Lilly
- Chris as Patch
- Lois as Stacy Jones
- Joe as Billy Twofeathers
- Stewie as PT Boomer