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When Luigi's brother Mario reveals a dark secret, Luigi must rise up with the help of an unlikely ally to stop his brother once and for all.
The film begins with Luigi drowning his sorrows in "Spaghetti on the rocks", only to be interrupted by his older brother's rude entrance. Luigi is surprised to see his brother, thinking the latter died in Zanzibar. Mario reveals the "Blue Shell Incident" was just a cover-up.
The film then cuts to a flashback of the supposed Blue Shell Incident, which features Mario and Luigi walking through an amusement park. Lasagna Ocelot, the alter ego of Garfield, stalks the duo and contacts a mysterious figure that "Operation Blue Shell Kill" is about to commence. He attempts to assassinate Luigi with his gun just as Mario ominously informs Luigi that he will go to hell before he dies. However, due to Lasagna Ocelot's laziness and it being a Monday, Lasagna misses the shot and it hits Mario instead. The cat retreats via a helicopter, seemingly out of ammo and is seemingly blown up by colliding into a random Toad in the sky. Luigi, upset over his brother's death, screams so loudly it can be heard in space.
It then cuts back to present day, where, through a series of pasta-related puns, Mario reveals he was faking his death all along and that it was his intention to kill Luigi. Grabbing his own gun, Mario intends to finish the job. A gunshot is heard and the screen cuts to black, only to reveal the gunshot was not fired by Mario, but by a new character: Duck Nukem. Duck Nukem reveals he witnessed the attempted assassination of Luigi and attempts to shoot Mario. Mario dodges, however, and the shot hits the Toad barkeep instead. Mario asks Nukem who sent him, but Duck Nukem's voice is replaced with unintelligble duck noises at the end, keeping it a mystery for both brothers.
Duck Nukem attempts to shoot Mario several times, but misses all his shots and empties his ammo. Mario makes a hasty escape through the window. Duck Nukem reveals he is on Luigi's side because Luigi fed him breadcrumbs when he was a duckling and because he thinks what Mario tried to do is despicable. The two chase Mario through a lenghty car chase scene.