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In tough conditions in Tuscany, where rain had left the pitch horribly cut up, the Giallorosse fell behind to Melania Martinovic's first-half finish - before Maegan Kelly turned home expertly inside the box midway through the second half.
Lindsey Thomas cut the deficit with 20 minutes remaining but, despite close calls for Elisa Bartoli and Vanessa Bernauer, were agonisingly unable to find an equaliser in the closing stages.
"Florentia were more determined, they had more courage and more spirit," Roma coach Betty Bavagnoli said afterwards.
"In the first half they won every tackle. In Serie A you will pay for that. The girls responded in the second half, but if they had played like that in the first half as well we would not have lost."
In the end, Bavagnoli's side found themselves on the wrong side of a hard-fought game, one blighted somewhat by the conditions and some dubious officiating - both of which prevented the game from really finding a rhythm.
Instead it often turned into an aggressive affair, with plenty of fouls and frustration for either side.
Both sides had their chances too, of course, but it was Florentia who did the better job of taking theirs - as Kelly and Martinovic both found the back of the net when it counted.
Roma, in contrast, came to rue a number of good openings that went begging - at least until Thomas took advantage of Manuela Giugliano's well-placed pass.
The Giallorosse dominated the closing stages, yet an equaliser remained frustratingly elusive.
The side are back in action next weekend, as Juventus come to Stadio Tre Fontane.
"That gives us a chance to get over the setback we suffered today," Bavagnoli added.
"We must believe in ourselves and focus on raising our game if we want to keep improving."
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