Fiorentina, who climbed to fourth on 13 points, took the lead from the softest of penalties for an alleged foul on Federico Chiesa in the 63rd minute.
Chiesa burst into the Atalanta area, slowed down and dribbled across the path of defender Rafael Toloi who did everything possible to avoid any contact, yet the Fiorentina player still went flying.
Surprisingly, the referee awarded a penalty without consulting the VAR system and Jordan Veretout converted. Cristiano Biraghi scored the second in the last minute.
As the final whistle went, a livid Gasperini began ranting angrily at his Fiorentina rival Pioli and there appeared to be a push between the pair before they were separated.
"Chiesa is a player who has improved enormously. Obviously, he may fall over if there is a physical contact but he doesn't go to ground at the slightest touch," said Pioli.
Genoa striker Krzysztof Piatek continued his remarkable start to his Serie A career by notching a brace in a 2-1 win at lowly Frosinone, who at least had the consolation of scoring their first goal of the season.
Piatek volleyed home in the 33rd minute and the Pole turned in a Christian Kouame cross from close range three minutes later, his eighth goal in six appearances since joining from Cracovia for a modest four million euros ($A6.4 million) in July.
Frosinone, who have one point, finally scored when Camillo Ciano converted a penalty three minutes before halftime after Nicolas Spolli fouled Stipe Perica.
Torino won 1-0 at Chievo after forward Simone Zaza came off the bench to grab a late winner, leaving the Flying Donkeys rooted to the bottom of the table with minus one point after having three deducted for accounting irregularities.
