The libretto for Mazeppa was written by Viktor Petrovich Burenin, based on Pushkin's poem Poltava. Tchaikovsky made changes to it, however, replacing Burenin's text with Pushkin's wherever possible, without losing the former's focus on the main character, Mazeppa, and without embracing the latter's greater historical scope.

The opera is cast is three acts and six scenes, its story set in early-eighteenth century Russia. Maria, daughter of the wealthy Cossack Kochubei, declares her love for the much-older Mazeppa. Andrei then announces his passionate love for her. Mazeppa asks of Kochubei his daughter's hand, but is denied. Kochubei's scheme to reveal Mazeppa's anti-tsarist intrigues to the Tsar backfires, landing him in a dungeon. Meanwhile, Mazeppa reveals his plans to Maria, who knows nothing of her father's imprisonment. Liubov, Maria's mother, apprises her of her father's plight. Maria hurries to save her father, but arrives moments after he is beheaded. During the Battle of Poltava, Andrei ambushes Mazeppa, who then shoots him. Maria comforts the dying Andrei, while Orlik drags Mazeppa off.

Tchaikovsky's music for the opera is often compelling, but is not one of his better scores. While Mazeppa is not a hero, neither is he quite the villain. Still, his character failed to inspire Tchaikovsky sufficiently, as Onegin did. Much of the better music in the opera is written for Mazeppa and for Maria. A deftly-used Leitmotif suggests his villainy at the beginning of the orchestral introduction and throughout the work. He also delivers a memorable arioso about his love for Maria in the first scene and sings beautifully about Ukrainian nights in Act II, scene 2. The music for both Maria and Mazeppa later in the same scene is quite moving, and it was reputedly this encounter that Tchaikovsky set to music ahead of anything else in the score. Perhaps the most beautiful and brilliantly ironic music in the entire opera is the closing number, when Maria delivers a lullaby to Andrei, deftly fashioned by Tchaikovsky on the same melody she uses in singing of Mazeppa as the future Tsar of Ukraine. Tchaikovsky employed folk music only in the first act, though some subsequent music may have folk roots. The opening girls' chorus is full of Russian-Ukrainian character, as is the hopak or Cossack dance that comes a little later. In the end, this is an opera with some masterful touches, but one lacking consistency.

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