The film opens with Mario playing his Family Computer. Suddenly, Mario witnesses a girl on the TV screen crying for help from the enemies attacking her and escapes by jumping out of the TV. After the enemies left, the girl reveals herself as Princess Peach. Just then, King Koopa appears and jumps out of the TV. Despite Mario's battling attempts, Bowser successfully captures Peach, and takes her back into the TV. Shortly afterwards, Mario discovers a small necklace that Peach left on the floor.
The next day while he and Luigi are working at a grocery store, they realize the necklace that Peach dropped becomes a matter of concern for Mario. Luigi then regards it as the "Visionary Jewel from the Country of Treasure". Soon, Kibidango, a small dog-like entity, wanders into the store and snatches the necklace from Mario, prompting he and Luigi to pursue him into the Mushroom Kingdom.
When the Mario Bros. arrive, an elderly hermit admits that Bowser has taken over the kingdom and is turning the helpless citizens into blocks. He also reveals that Friday the 13th is the marriage between King Koopa and Peach. The mushroom advises the Mario Bros. to find the three powers; the mushroom of strength, the flower of courage, and the yellow star of invincibility, in order to defeat Bowser and save Peach.
After a long journey and overcoming the many perilous obstacles, the Mario Bros. eventually acquire all the three powers by Friday the 13th. That night, Mario arrives at King Koopa's castle just as the wedding of King Koopa and Peach started. With the help of the three powers, Mario successfully defeats Bowser, and his magic is destroyed, turning the Mushroom Kingdom back to normal. When Mario gives Peach's necklace back to her, Kibidango arrives in his true form, Prince Haru of the Flower Kingdom. Haru admits that it was King Koopa who turned him into Kibidango. He also reveals to the Mario Bros. that he and Peach are betrothed to each other and are fated to marry no matter what. Heartbroken, Mario decides to leave the Mushroom Kingdom to allow Peach to marry Haru as previously planned. After a tearful farewell from Peach, the Mario Bros. are seen leaving the Mushroom Kingdom, making their long journey home as the credits roll. In the first post-credits scene, the Mario Bros. wave good-bye as they enter a pipe that says, "Grouper Productions".
In the second post-credits scene, a customer who appeared earlier in the film visits the grocery store, only to discover that Bowser and his minions are now working as punishment. She is visibly overjoyed with better service and cries out as the background changes to blue and the words "Game Over" appear while the Super Mario Bros. "game over" music plays.
The film's soundtrack consists of these songs, among others:Adieu My Love (アヂュウマイラブ, Adyū Mai Rabu)
Crystal Ball (水晶玉, Suishō Tama)
Doki-Doki Do It! - Rock'n'roll
Doki-Doki Do It!
Additionally, the film uses music and sound effects from the video game Super Mario Bros. An LP was released.
In 1986, Mario was already popular in Japan so Grouper Productions collaborated with Nintendo to produce a Mario movie. To advertise the anime, they released Mario phone cards, watches, rice containers, ramen noodles, a manga, an art book, three riddle books, a picture book, and a limited edition soundtrack, the last one only having fifty copies produced. On July 20, 1986, the anime came out in theaters across Japan. How much it grossed remains unknown. VAP Video later released the film on retail VHS and Betamax cassettes. All merchandise associated with the film is extremely rare, with the VHS tape being credited as one of the rarest pieces of Mario merchandise ever made.