Barcelona warmed up for the game by thrashing Sporting Gijon 4-0 over the weekend and were boosted by the news that both their main rivals for second place in the Spanish League and the leaders, Real Madrid, were held to draws.
But seven points still separate Barcelona from their arch-rivals and their coach Bobby Robson knows that his main chance of serving out the second year of his contract lies in taking a European title.
Barcelona won the Cup-Winners' Cup in 1979, 1982 and 1989 but Fiorentina are far from overawed. Their Argentinian striker Gabriel Batistuta said: "I've never felt inferior to anyone. Sure, the Catalan side has 22 great players and lots of international experience but the real Fiorentina can beat any side."
The Fiorentina coach, Claudio Ranieri, believes it would be a major tactical error for his side to concentrate all their attention on Barcelona's Brazilian striker, Ronaldo.
''That would be equivalent to allowing a free hand to all the other nine players, all equally useful," he said.
Fiorentina, who were unlucky in a 0-0 draw with Internazionale on Saturday, are ninth in Serie A, 17 points behind the league leaders, Juventus.
That lowly league standing reflects the extent to which the Florence club have based their whole season on their European campaign. They will be handicapped by the absence of two first-choice players. The former Everton winger Andrei Kanchelskis is cup-tied while the former Arsenal midfielder Stefan Schwarz is suspended.
Although Ranieri was giving nothing away about his plans, he may be tempted to play a cautious 4-5-1 formation to compensate for the loss of Schwarz's covering work and the extra width added by Kanchelskis.
Sandro Cois, Emiliano Bigica, Anselmo Robbiati and the Brazilian Luis Oliveira could all line up in midfield alongside Rui Costa, leaving Batistuta on his own up front.
As for Ronaldo, he may be marked in defence by Lorenzo Amoruso and in midfield by Cois. Robson will be without the key midfielders Ivan de la Pena and Josep Guardiola, both of whom are suspended.
Guardiola and De la Pena tend to supply the ideas, while injuries have put paid to the two Barcelona men best known for their commitment and pace - the overlapping defender Sergi and the utility man Luis Enrique.