The match started in open fashion and it was the Italians who claimed the first attack, Milan Badelj latching on to a loose ball outside the box and blazing a shot over the bar.
Fiorentina felt they should have been awarded a penalty in the 13th minute as Joaqin's strike appeared to hit Yevhen Khacheridi on the arm, but the referee saw nothing wrong with the defender's interception.
Mohamed Salah proved a handful for the Kiev defence in the first period and he almost lined up Mario Gomez for the opening goal in the 17th minute, the German smashing into the side-netting from a tight angle under pressure from his marker.
Kiev had a chance to open the scoring at the other end in the 25th minute as Jeremain Lens combined with Andriy Yarmolenko before testing Neto in the Fiorentina goal with a low strike that took a deflection on its way into the keeper's arms.
The Viola came close again just after the half-hour mark as Matias Fernandez played a one-two with Gomez before curling a shot just wide of the far post from outside the area.
The Ukrainians took the lead against the run of play in the 36th minute as Lens hit from outside the box and beat Neto with the help of a wicked deflection that left the Brazilian stranded.
Fiorentina had the chance to level things up minutes before the break when they won a free kick in a dangerous position, but Fernandez's strike ricocheted behind and the teams went into half time at 1-0.
Kiev were quick to make a statement of intent in the opening stages of the second half, with Serhiy Rybalka running at the Viola defence and flashing a shot wide within 30 seconds of the restart.
The visitors had another penalty shout waved away just before the hour mark as Borja Valero went down in the box under a heavy challenge from Domagoj Vida, but the referee dismissed the Fiorentina protests.
Vincenzo Montella's side struggled to gain a foothold in the game as the second half wore on, but tested Kiev keeper Oleksandr Shovkovskiy in the 74th minute when Salah exchanged passes with Nenad Tomovic and bent an effort towards goal.
They were left ruing their misfortune moments later as a glancing header from Valero came back off the outside of the post, the midfielder doing well to meet the ball from a tight angle.
However, Fiorentina's pressure paid off in the second minute of stoppage time as Babacar met a looping cross with an overhead kick and netted a vital away goal for his side.