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The Giallorosse, looking for revenge after the midweek cup exit against the same opposition, put up a determined fight at the Stadio Bozzi - only to see a last-minute penalty condemn the side to defeat.
Luisa Pugnali cancelled out Ilaria Mauro's opener by rounding off a fine team move just before half-time - although in truth Betty Bavagnoli's side did not create a great number of opportunities themselves.
But they defended well and it was only a harsh penalty decision that allowed Valery Vigilucci to decide the game from 12 yards at the death.
"I'm proud of the girls," Bavagnoli said afterwards. "They showed the quality they have and, equally importantly, listened to what we had to say after Wednesday's defeat.
"With the mentality we have, and the investment still to be made in the future, we can achieve great things. We need to make the right moves to keep building on what we've done this season."
Roma had already clinched fourth spot in Serie A prior to the match, although the win did ensure Fiorentina finished second, behind champions Juventus, and will play in next season's Women's Champions League.
The Giallorosse finished with 36 points from their 22 games - winning 11 and drawing three.
"Fiorentina aren't a side that need to be gifted things, yet this match was still decided by individual moments," defender Federica Di Criscio added.
"We finish the season with our heads high, we've shown that even though we are a young squad we can have a big impact in this league."
See more photos in the gallery below.
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