At its core, the Dr. Mario setting and gameplay is unchanged from previous releases. Germs of three colors fill the playfield. Pills with two halves of random color are dropped, and the player tries to align them with germs of the same color. Four or more of the same color in a row (any combination of germs and pill halves) and all in the line will disappear. Gameplay continues until the player clears the field of germs, or pills pile to the top.

The game features offline and online one-on-one multiplayer modes. There's Classic versus mode with the same win conditions as the single-player mode; as well as Flash, where the winner is the first one to eliminate their flashing germs. Online play can be against random players or those on a friends list. A friend with a Wii but not a copy of Dr. Mario Online Rx can be sent an invitation to download a demo version that's only usable to play against people with the full copy.

A variant of Dr. Mario called Virus Buster is available for offline play, 1-4 players cooperative. First seen in Brain Age 2 as a stylus-controlled version of Dr. Mario, now the Wii Remote's pointer is used to grab and move pills, with the B button used to rotate them. Pills and germs display much larger in this mode.