Teenage Mutant Ninja Turtles: Mutants in Manhattan is a 3D brawler featuring the four turtles Donatello, Leonardo, Michelangelo and Raphael who are all playable. The game takes place in New York where the Foot gang is involved with criminal activities. The turtles intervene, but their journalist friend April Ryan thinks it is the precursor to something larger. Many familiar villains return such as Bebop, Krang, Rocksteady, Shredder, Slash and Wingnut. In single-player it is possible to switch between the four turtles at any moment and the remaining three are always controlled by the AI. Cooperative gameplay is available for four players through online multiplayer only.

In nine stages the turtles need to take out groups of opponents and bosses using a variety of moves that can be combined into combos. Each turtle has a different fighting style and a unique ability, for instance slowing down time. General items include health-restoring pizza, deployable turrets, and bombs. The environment can be scanned to tag enemies and movement includes parkour-like actions to clamber up the sides of buildings as well as parachutes to glide over rooftops. Along the way green orbs can be picked up for Battle Points used to upgrade abilities. There is an experience system and a number of passive skills earned through high ranks in missions. Downed turtles can be revived, but when this is not done in time they are sent back to their lair and participate in a mini-game involving eating pizza to return. Next to fighting tasks include disarming bombs, destroying objects and infiltrating hideouts.