The defeat left Napoli four points behind leaders Juventus with three matches to play, only a week after re-igniting the title race with a last-minute win away to the Turin side.
Juventus won 3-2 at Inter Milan on Saturday after scoring twice in the last five minutes.
Koulibaly was dismissed for felling Simeone as he was about to shoot. The referee at first gave Fiorentina a penalty and handed the Senegalese defender a yellow card, but changed that to a free kick for the home side and a red card for Koulibaly after reviewing the incident with the video assistant referee (VAR).
(Writing by Brian Homewood in Bern, editing by Pritha Sarkar)
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