Police said the fire started around 02:00 local time (00:00 GMT) during a performance at the Contrast nightclub, - BBC reported.
Three men and three women aged in their twenties died trying to escape, police official Predrag Maric said. Some 300-350 people had been in the club.
Police said the likely cause was an electrical fault. The club's owner and two others have been arrested.
Mr Maric said the victims died from smoke inhalation.
"One of them was just half a metre from an exit," he said. "There must have been huge panic, they all rushed to get out."
Investigative judge Ana Stamenic told Reuters news agency that prosecutors were considering charges of "causing general danger" in relation to the blaze. She said all six victims had been identified.
Novi Sad Mayor Igor Pavlicic said the nightclub had been regularly checked for safety compliance and had recently carried out a fire drill.
The mayor declared three days of mourning from Monday.
"This is a great tragedy," he said. "The wounds of past tragedies in Novi Sad have yet to heal, and now we have another."
In 2007, three people died in a hotel fire in the city and a year earlier a cafe fire left eight dead.