The Giallorossi felt aggrieved at an early penalty awarded against them - after a VAR check - as Nico Gonzalez stepped up and converted from the spot.
Giacomo Bonaventura then slotted home five minutes later to double the advantage for the hosts, who were able to see out the remainder of the contest.
The result complicates the European somewhat for Jose Mourinho's side: they now sit level on points with both Fiorentina and Atalanta with two games remaining in Serie A.
Roma are back in action on Saturday - they face relegation-threatened Venezia at the Stadio Olimpico.
FIORENTINA 2-0 ROMA
GOALSCORERS: Gonzalez (5' p.), Bonaventura (11')
FIORENTINA: Terracciano; Venuti, Milenković, Igor, Biraghi (Terzic); Bonaventura (Maleh), Amrabat, Duncan; González (Callejon), Cabral (Piatek), Ikoné (Saponara)
ROMA (3-5-2): Rui Patricio; Mancini, Smalling, Ibanez; Karsdorp (Spinazzola), Veretout, Cristante, Sergio Oliveira (Zaniolo), Zalewski (El Shaarawy); Pellegrini (Perez), Abraham (Shomurodov)
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