Fielding an almost entirely different starting XI from the Community Shield defeat to Arsenal, the Premier League champions struggled to create many clear-cut chances at Stamford Bridge.
Loic Remy and Gary Cahill spurned Chelsea’s best opportunities after Gonzalo Rodriguez had broken the deadlock from close range in the 34th minute.
Chelsea will now turn their attentions to their opening fixture of the 2015/16 Premier League season at home to Swansea on Saturday evening.
The Blues made 10 changes to the side beaten 1-0 by Arsenal at the weekend, with skipper John Terry the only player to keep his place, while Radamel Falcao, Asmir Begovic, Bertrand Traore and Ola Aina made their home debuts.
Begovic was called into action early to pull off a good save from Matias Fernandez’s first-time shot from the edge of the area.
Chelsea then began to take control, Ruben Loftus-Cheek heading over and seeing a shot blocked and Aina having an effort deflected over.
But, after Matias Vecino had thundered a shot against the bar for the visitors and Begovic had parried a curling attempt by Khouma Babacar, Fiorentina struck.
A cross from the right was chested down by Marcos Alonso and although his left-foot effort was blocked at the near post by Begovic, the ball squirted out to Rodriguez, who bundled home.
Beginning at half-time, Chelsea made nine changes in the second half, with Eden Hazard, Cesc Fabregas, Cahill and Nemanja Matic among the first-team stalwarts to enter the fray.
Fiorentina, meanwhile, sent on nine substitutes, including Italy striker Giuseppe Rossi, who missed the entirety of the 2014-15 season following the third serious knee injury of his career.
Substitute Josip Ilicic and Ante Rebic threatened for Fiorentina and although Chelsea finished the game in the ascendancy, they could not find an equaliser.
Cahill saw a pair of headers saved by Luigi Sepe and Remy, who replaced Falcao on the hour, shot wastefully wide after cutting inside Alonso.