Mario and Luigi find themselves in a strange dystopian parallel world where a human-like race evolved from dinosaurs rather than the mammalian ancestry of true humans in a Manhattan-like city. Sixty-five million years ago, a meteorite crashed into the Earth and in doing so ripped the universe into two parallel dimensions. All the surviving dinosaurs of the time crossed over into this new realm. Iggy and Spike turn out to be henchmen (and cousins) of the other world's germophobic and obsessive dictator, King Koopa, descended from the T-Rex. However, the two have failed to also bring Daisy's rock, a meteorite fragment which Koopa is trying to get in order to merge his world with the real world that separated from Koopa's world during the meteor strike. It turns out that Daisy is the Princess of the other dimension but when Koopa overthrew Daisy's father (and turned him into fungus), Daisy's mother took her to New York using the inter-dimensional portal. The portal was then destroyed, killing Daisy's mother in the process; but when Scapelli was blasting at the cave, the portal was reopened. When Koopa hears about the re-opening of the portal, he sends Spike and Iggy to find Daisy and the rock to merge the dimensions and make Koopa dictator of both worlds. Spike and Iggy, however, who had grown more intelligent after being subjected to one of Koopa's experiments, decide to turn on Koopa and join forces with Mario and Luigi. Koopa thinks only Daisy can merge the worlds, but Mario and Luigi were just in the wrong place at the wrong time. Daisy is eventually rescued by the plumbers, along with the help of Toad, a man punished by Koopa for performing music protesting his reign, then condemned to be "devolved" into a Goomba.
Eventually, the two worlds merge and Koopa inadvertently devolves Scapelli into a primate while aiming for Mario, but Luigi and Daisy take back the rock and the worlds separate again. Mario confronts Koopa and eventually wins when he and Luigi devolve him, transforming him into a ferocious, semi-humanoid Tyrannosaurus. Koopa then leaps out for a final attack but Mario and Luigi destroy him by devolving him further into primeval slime. Daisy's father, the king named Bowser, turns back to normal and reclaims control over the kingdom stating he loves those plumbers. The citizens destroy anything involving Koopa. As the brothers return home, Luigi and Daisy admit their love for one another but Daisy cannot return to New York until the damage caused by Koopa is reversed and she spends more time with her father. Mario rephrases Daisy's words to Luigi but he does not care. A deeply hurt and saddened Luigi kisses her goodbye and the two brothers return to New York, while Daisy watches them leave. About three weeks later, Daisy returns for Mario and Luigi's help in fighting more villains. Meanwhile, Mario and Luigi's story is televised, giving them the name; "Super Mario Bros".