Rossi, usually one of the calmest coaches in the histrionic Italian league, lost his cool when substituting Serbian midfielder Adem Ljajic in the first half of the 2-2 home draw with relegated Novara on Wednesday.
The player ironically applauded the coach as he entered the dugout and Rossi grabbed Ljajic before landing two blows while scrabbling around on the ground.
"The first team squad for the last two games of the season has been entrusted to Vincenzo Guerini," the not yet safe Serie A side said in a statement, referring to Rossi's actions as a "very serious episode".
"Fiorentina also announce we have started disciplinary proceedings against Adem Ljajic."
Fiorentina travel to third-bottom Lecce on Saturday needing a point to secure safety after a woeful season for one of Italy's most recognisable clubs.