Their 1-0 home win over already-qualified Olympique Lyon on Tuesday sent the Serie A side into the knockout round and eliminated five-times winners Liverpool.
Coach Cesare Prandelli says there is more to come from his side.
"This qualification represents something huge," he told reporters after Juan Vargas's first-half penalty sealed the win.
"This game has given us the confidence to be challengers at a European level. I am satisfied but I like to think you can always do better."
The Florence club, who can keep top spot in Group E with a second win over Liverpool next month, last got out of the first group stage in 1999 when Argentina striker Gabriel Batistuta was in his prime.
Bankruptcy and relegation followed before a Florentine renaissance bankrolled by the Della Valle family, although their commitment to the club was questioned in September when Andrea nominally quit as president after becoming disillusioned.
Further Champions League success could again whet their appetite, especially given Fiorentina won on Tuesday without Adrian Mutu, hot young prospect Stevan Jovetic and top defender Alessandro Gamberini.
Italian media hailed their progression and talked up Prandelli as a future Italy boss, while European underachievers Inter Milan were again lamented for a weak 2-0 loss at Barcelona, which complicates their hopes of reaching the last 16.
