Lorik Cana's early strike was enough to claim all three points for Lazio, who were knocked out of the Europa League after an aggregate 4-3 defeat to Ludogorets on Thursday.
Edy Reja's side moved up two places into eighth, while Fiorentina remain in fourth in the Serie A standings.
Albania international Cana scored with the first chance of the game after just five minutes as he slotted home with a half-volley at the far post following a corner for the visitors.
Two minutes later Antonio Candreva could have doubled Lazio's lead but his effort from the right wing was saved by Fiorentina goalkeeper Norberto Neto.
The home side hit back with a series of attempts to level the score midway through the first half.
Firstly, Alessandro Matri failed to tap in Nenad Tomovic's cross, before the Serbia defender had a chance himself to net an equaliser before firing the ball over the bar from outside the 18-yard box.
Matri had another go but his header from a corner was also over the bar, and Manuel Pasqual set up Matias Fernandez with a cross from the left wing but his close-range header was off target.
Lazio almost netted their second just before the break as Candreva picked out Abdoulay Konko at the far post, but his effort hit the bar.
The visitors started the second half strongest before Fiorentina fought back with a chance from Joaquin Sanchez, but his shot flew high over the bar from outside the box.
Candreva started a counter-attack for Lazio minutes later and found Brayan Perea inside the box but he was denied by Gonzalo Rodriguez near the goal line.
Fiorentina had the chance to net a leveller late in the match with two great chances from Anderson and one from Fernandez but despite their efforts, Lazio went home with maximum points.