Carlo Ancelotti's Napoli had fallen to their first defeat of the season before the international break 3-0 to Sampdoria in Genoa.
But Insigne broke through after 79 minutes in the San Paolo Stadium, whipping in his 50th Serie A goal following a cross from substitute Arkadiusz Milik.
It handed Fiorentina -- who ended Napoli's title hopes last April -- their first defeat of the season.
"In Genoa we weren't ill, we just had a little cold," said Ancelotti.
"Today we were more balanced and effective. We played the game we wanted today even if it wasn't an exceptional level.
"It's pleasant, exciting and wonderful to be back in the Champions League. We've got a tough group and will try to get a good start."
Napoli move up to second with nine points, equal with champions Juventus who host Sassuolo on Sunday with Cristiano Ronaldo still waiting for his first goal.