David Moyes' side had been unbeaten in Europe this season after an impressive series of displays in the group stage and first knockout round.
However, they were strangely out-of-sorts in difficult conditions in Florence and went down to a comprehensive defeat at the hands of a side currently riding high in fourth place in their domestic league.
Moyes had named an unchanged starting line-up for the third successive match, which meant fit-again midfielder Mikel Arteta was only named on the bench.
But even the second-half arrival of the Spanish midfielder following a recent stomach problem could not pep up a lacklustre Toffees display, in which they failed to significantly test home goalkeeper Sebastien Frey throughout the entire 90 minutes.
Veteran striker Christian Vieri scuffed a couple of early chances wide for the home side before Manuel Pasqual went close with a well-hit shot after arriving late into the penalty area midway through the first period.
Everton were struggling to retain possession with the disappointing Yakubu largely anonymous as a lone striker and it was no surprise when the home side continued to dominate the match.
Tim Howard saved the Toffees as he deflected a Vieri effort from close range out for a corner somewhat fortuitously in the 42nd minute.
The pattern of the game continued after the interval as Howard palmed a wicked bouncing shot from 25 yards by Riccardo Montolivo around his post in the 49th minute.
The American keeper then produced an excellent double save to keep out first Vieri and then Zdravko Kuzmanovic from the edge of the box as Fiorentina kept up the pressure.
However, any hopes the Toffees had of riding the storm were wrecked on 70 minutes when the home side finally found a way thorough. Tomas Ujfalusi teed up Kuzmanovic and he found the net with a quality low right-footed curling shot from the edge of the box.
Ruthless Fiorentina then doubled the advantage 11 minutes later when Martin Jorgensen flicked the ball back to the onrushing Montolivo, who lashed a crisp low volley past the helpless Howard from 12 yards.
It could have been worse for the Toffees had substitute Mario Alberto Santana not guided an angled shot marginally wide of the far post three minutes from time.
But Everton now face an uphill task to reach the last eight of a competition in which they were considered among the favourites prior to this below-par showing in Italy.