After Mario and Luigi save Princess Peach, the Mushroom Retainers and the Mushroom Kingdom from the evil King Koopa's evil spell, Bowser kidnaps her with the Mushroom Retainers and invades the Mushroom Kingdom again. This time, Mario and Luigi have to travel in many different and dangerous worlds of the kingdom to rescue the princess and the inhabitants from the False Bowsers. Though the lands seem very familiar to Mario and Luigi, they are much more dangerous and inhabited by more enemies than they had ever experienced before. Despite that, the Mario Bros. go through the nine new lands of the Mushroom Kingdom, fighting the Koopa Troop and False Bowsers in each castles when freeing the Mushroom Retainers until they reach the real Bowser and Princess Peach. After that, they defeat him and rescue the fair princess once more.

The original version of this game was released on the FDS in 1986.