The Giallorossi go into their all-Italian tie at the Stadio Olimpico having endured a disappointing spell in Serie A.
Roma have won just one and drawn eight of their last 10 league games to drop 14 points behind leaders Juventus.
Monday’s 2-0 defeat to Sampdoria was Roma’s first league loss at home since May 2014 and put further pressure on coach Rudi Garcia heading into Thursday’s match.
“We are going through a negative spell,” Roma playmaker Miralem Pjanic told the Italian press. “It’s a difficult time for the team and for our fans.
“But I hope that after our defeat to Sampdoria, results will come our way.
“I am certain on Thursday we will win and go through to the quarter-finals.”
A goalless result will be enough for Roma to progress to the next round after Seydou Keita netted an all-important equaliser to hand the Giallorossi a precious away goal in their 1-1 first-leg draw.
Fiorentina travel to the Italian capital in buoyant mood.
The Viola came from behind to beat AC Milan 2-1 on Monday and climb to fifth in Serie A, five points adrift of Roma.
“This win is a huge boost in confidence,” Fiorentina winger Joaquin said. “We showed great mental strength.
“We know the important of Thursday’s game and we just have to continue to do well and take it one game at a time.”
Fiorentina, coached by former Roma striker Vincenzo Montella, are unbeaten in 10 European away encounters and have scored in all but one of those games.
The Viola beat Thursday’s hosts 2-0 at the Stadio Olimpico last month to progress to the Coppa Italia semi-finals.
German forward Mario Gomez, who scored both goals in that game, will not feature on Thursday as he is sidelined through injury.
Mohamed Salah, who has scored four goals in eight appearances since joining Fiorentina on loan from Chelsea in January, will partner Khouma Babacar in attack.
Roma are without suspended midfielder Radja Nainggolan but are bolstered by the return of midfielder Daniele De Rossi and captain Francesco Totti from injury.