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Mario (Bob Hoskins) and Luigi Mario (John Leguizamo) are two Italian American plumbers living in Brooklyn, New York. The brothers are being driven out of business by the mafia-like Scapelli Construction Company, led by contractor Anthony Scapelli (Gianni Russo). Mario is the elder of the two brothers, being close to the late 30s, and Luigi is about in his early-mid 20s. Luigi falls in love with an orphaned NYU student, Daisy (Samantha Mathis), who is digging under the Brooklyn Bridge for dinosaur bones. After a date, she takes Luigi to the dig and witnesses Scapelli's men (who, along with Scapelli himself, had previously threatened her to end her research on that specific piece of land for their own interests) sabotage it by leaving the water-pipes open. Luigi tries to stop it but he does not have his tools on him so he can not fix it. They rush back to his apartment where they inform Mario about the incident. The three go back to the flooding and the Mario Bros. manage to fix it but are knocked out by two strange characters, Iggy (Fisher Stevens) and Spike (Richard Edson). The two crooks kidnap Daisy.

Mario and Luigi awaken a minute later and head deeper into the caves following Daisy's screaming and discover an interdimensional portal through which Mario and Luigi follow Daisy. They find themselves in a strange dystopian parallel world where a human-like race evolved from dinosaurs rather than the mammalian ancestry of true humans. 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 (Dennis Hopper), 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.

Eventually, the two worlds merge, during which Scapelli gets his comeuppance when Koopa devolves him into a primate by mistake while aiming for Mario, but Luigi and Daisy take back the rock and the worlds separate. Mario gains his courage and fights Koopa and eventually wins when he and Luigi devolve him, transforming him into a ferocious, semi-humanoid Tyrannosaurus. Koopa then leaps out for a last attack but Mario and Luigi devolve him into primeval slime, destroying him. Daisy's father (Lance Henriksen) 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, along with Yoshi and Toad (Mojo Nixon), watch 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 their name "Super Mario Bros."

In a post-credits scene, two Nintendo executives talk about a video game based on their adventures but they are asking Iggy and Spike instead of the Super Mario Bros. and they decide on a title called "The Super Koopa Cousins".
(C) Wikipedia