The first and last parts of each episode were live action and showed Mario (played by "Captain" Lou Albano) and Luigi (Danny Wells) living in Brooklyn, where they would often be visited by celebrity guest stars. Some of them were popular TV stars, such as Nedra Volz, Norman Fell, Donna Douglas, Eve Plumb, Vanna White, Jim Lange, Danica McKellar, Nicole Eggert, Clare Carey and Brian Bonsall or professional athletes such as Lyle Alzado, Magic Johnson, Roddy Piper and Sgt. Slaughter. Occasionally, the main actors would be playing guest stars themselves, forcing their regular characters to leave when it came time for their other characters to show up. In the fifth episode, Ernie Hudson appeared as a Ghostbuster using his own name rather than Winston Zeddemore.

The second part of each episode was a 13-minute cartoon based loosely on the Super Mario Bros. and Super Mario Bros. 2 video games, where Mario, Luigi, Princess Toadstool (as Peach was called in North America until the release of Yoshi's Safari), and her loyal mushroom retainer Toad battle against the reptilian villain King Koopa,a.k.a. Bowser, often in a movie or pop-culture parody. Getting into the spirit of these parodies, Koopa usually had a different outfit for each one. Wart, the main antagonist of the second game, was never in any of the episodes, yet most of his minions appeared in the show. The cartoons were meant to take place after the timeline of the Super Mario Bros. game where the Princess was rescued. It was revealed that the Mario Brothers accidentally were warped into the Mushroom Kingdom while working on a bathtub drain in Brooklyn, NY, and in appearing presented a challenge to King Koopa's plots to overtake the kingdom. The brothers and Toad had to protect the Princess, as they believed she had the power to save her people as well as to get the Mario Brothers back to Brooklyn. It was never explained whether the live action segments were meant to be the Mario Brothers before they found the Mushroom Kingdom, or if they had successfully made it back to Brooklyn and returned to operating their plumbing business as before.

The Super Mario Bros. cartoon was shown on Mondays through Thursdays. On Fridays, the show would air the Legend of Zelda cartoons based on the game of the same name, in which the elf-like hero Link and Princess Zelda fight against the forces of the evil wizard Ganon. Scenes from the episode were shown during the live-action segments on the preceding days as sneak previews.

During the summer of 1990, Club Mario replaced the Mario Bros. live-action segments. This featured rock and roll and Mario obsessed teenagers (Chris Coombs, Michael Rawlins, and Victoria Delany) goofing around, and in at least one episode, running around the DiC studios and harassing Andy Heyward. Coombs and Delany played Siblings Tommy and Tammy Treehugger. An additional added segment was a 1-2 minute viewing of "Spaced Out Theater", which was edited from the TV series, Photon.

The show ran from September 4 to December 1, 1989. DVD box sets of SMBSS were released on March 28, 2006 (Volume 1) and October 31, 2006 (Volume 2) by Shout! Factory.

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