Attempting to sync another controller will fail. When switching the game to or from this mode, all controllers currently synced to the Nintendo Switch will turn off.

Joy-Con (L) is always assigned to Player 1, and Joy-Con (R) is always assigned to Player 2. This mode is only playable using two Joy-Con controllers. This mode also allows Player 2 to fire even during Mine Cart sections and to use the pointer when inside it, which is usually not possible. They cannot be used to activate switches, enter Clear Pipes, break Rock Blocks, or damage bosses. Red turnips can be used to obtain collectibles (except Super Gems and Power Stars), defeat enemies, or break small blocks. Player 2 also gains the ability to fire red turnips towards wherever the pointer is located. In Assist Mode, Player 1 assumes the normal role of Captain Toad or Toadette, except for the change that Player 2 now exclusively controls the pointer. Both of these modes are only available in the Nintendo Switch's TV / Tabletop mode. In the Nintendo Switch version of the game, two types of cooperative two player modes are available in every level. Catch Pixel Toad - (only on Tabletop and TV mode).Play Hide and Seek with Pixel Toad - (only on menu).Interact with object and enemies - (only on Tabletop and TV mode).Use pointer - (move) (only on Tabletop and TV mode).Run/pluck/throw - / / / (only on Handheld mode).Play Hide and Seek with Pixel Toad - Tap Pixel Toad on touch screen (only in level select menu).Use amiibo - Tap amiibo button on touch screen (only in level select menu).Tilt - Gyroscope (if enabled in-game) / Touch Screen / C-Stick.

The Nintendo Switch version of the game can be played without the touch screen in Tabletop or TV mode and with the touch screen in Handheld mode.

Any other amiibo can give Captain Toad 1-Up Mushrooms. In addition, the Wedding Mario, Peach, or Bowser amiibo unlock the Super Mario Odyssey-themed courses early in the game. The Toad amiibo now grants the player invincibility for a level, similar to the effects when scanning a Mario amiibo in Super Mario Odyssey, rather than to play Hide & Seek, which is instead unlocked by completing a level. Like the Wii U version of the game, this game is compatible with amiibo.

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