The game takes place after Mario and Luigi: Bowser's Inside Story. Fawful sits down in a forest in the Bean Bean Kingdom. He plans to make a species that can destroy Mario, Luigi and Bowser. He flies to Bowser's Castle and goes into the freezer room and sees 15 frozen shroobs. He laughs an evil laugh while pulling down a lever to thaw out the shroobs. The screen fades to Mario and Luigi's house. Stuffwell was sitting against a wall. Mario bought a new HD TV to put on a wall. Luigi had the Poltergust 3000 by his bed and the F.L.U.D.D. by Mario's bed. Mario and Luigi were sitting in their chairs by their new fireplace. Toadsworth runs in the house. "Th... th... the shroobs are bb... back!" Toadsworth screams. He faints. Mario and Luigi jump out of their chairs and run to Peach's Castle.
Baby Mario (X)
Baby Luigi (Y)
Bowser Jr. (L)
Shroob Mario and Shroob Luigi
Theme: New Super Mario Bros. and New Super Mario Bros. Wii Overworld Theme mixed
Koopa Bros. Theme (optional battle with Koopa Bros.)
Shroob Attack Theme
Yoshi's Island (DS): Kamek reminds Mario, Luigi, Baby Mario, Baby Luigi and Yoshi of the time Baby Mario and Yoshi saved Baby Luigi
Mario and Luigi: Partners in Time: Shroobs return
Mario and Luigi: Bowser's Inside Story: You can play as Bowser again and Fawful returns
Mario and Luigi: Superstar Saga: Cackletta returns except in a DNA transformed Shroob form
Super Mario Galaxy: Good Egg Galaxy is seen in the background when the Shroob UFOs destroy all the planets and galaxies
Metroid series: Mother Brain is a boss.
Super Mario World and Sonic the Hedgehog series: The blue hedgehog is an enemy
Paper Mario: The Koopa Bros. are an optional boss
Super Paper Mario: Count Bleck is in the Mushroom Kingdom Museum. If you talk to him he will laugh "heh, heh, heh, bleck"