Annamaria Serturini and Andrine Hegerberg scored the goals for Betty Bavagnoli's side, who secure Roma's first win against one of Italian women's footballs recognised 'big' sides in the process.
It was Serturini who effectively changed the course of the game, as last season's top goalscorer produced an early goal of the season with a 30-yard right-footed rocket that arrowed into the top corner.
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In truth Fiorentina had perhaps enjoyed the better of the match before that point - Camelia Ceasar saving a penalty from Tatiana Bonetti - but, as Roma went into the break ahead, they came out for the second half in determined mood.
A red card for midfielder Stephanie Breitner after a poor foul on Agnese Bonfantini was evidence of how frustrated La Viola were becoming - and moments after that it was Hegerberg who clinched the game.
Scoring her first goal for the club, the Norwegian midfielder reacted quickest inside the box to slide home after a mistake from the Fiorentina goalkeeper.
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Roma saw out the rest of the game, securing an eye-catching result and - perhaps more significantly - underlining their character, as they bounced back from last weekend's disappointing loss against AC Milan.
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