In a battle of out-of form sides, Nicolas Gonzalez converted a fifth minute penalty awarded after he was mowed down by Rick Karsdorp.
Six minutes later the Roma defence allowed Giacomo Bonaventura the time to bury a shot in the bottom corner.
Roma coach Jose Mourinho picked nine of the starting 11 who drew in Leicester on Thursday to reach the Europa League final but the team stretched their winless league run to five games.
Fiorentina ended a run of four games without a victory and overtook Atalanta to climb into seventh, which this season will bring qualification for the Europa Conference League.
Both are level on points with Roma who trail neighbours Lazio by three points.
The two Rome clubs occupy the two Europa League places. (AFP)