"The main problem is knowing who is actually refereeing the match and sometimes I have the impression that the match is being refereed by the video assistants," he said during a conference organised by the Italian federation (FIGC) on VAR.
"I can accept mistakes by (Piero) Giacomelli and (Gianluca) Rocchi, but I don't accept mistakes by the VAR," said Ancelotti, referring to two of Italy's top referees.
The VAR system is being used in Serie A for the third season and it continues to cause controversy, as much as for incidents which are not reviewed as for the ones which are.
Ancelotti was furious about an incident during a match against Atalanta when his side had a penalty appeal waved away. Napoli were leading 2-1 when the incident happened and Atlanta went straight down the other end and equalised.
Referee Giacomelli decided not to review the incident on the pitch side monitor, the game finished 2-2 and it has proved a turning point for Napoli as they have not won any of their three games since.
"Without beating about the bush, did (the referee) make a mistake in Napoli-Atalanta?" Ancelotti asked Nicola Rizzoli, who is charge of the Serie A referees.
Rizzoli, a former referee who took charge of the 2014, World Cup final, replied that the officials should have looked at the incident.
Ancelotti jokingly replied: "Good, I am happy now.. ..I can leave."
Rizzoli said there had been 52 VAR interventions so far this season in 12 rounds of matches, which he admitted was too many although he added that changes to the handball rule had complicated things.
"This year, VAR has intervened once every two games, whereas at the same point last season it was one every four," he said. "It means we are not refereeing well although that is also because the rules have changed."