Orlando Police said officers entered the Pulse Club about three hours after the shooting unfolded - and killed the attacker, who had taken hostages.
They called it an act of terrorism, but said they did not yet know if it was domestic or international.
Relatives have been gathering at local hospitals desperate for news.
A man who had been inside the club earlier described a situation of chaos outside as the number of casualties became apparent.
"There were just bodies everywhere," Christopher Hansen said. "In the parking lot, they were tagging them - red, yellow - so they knew who to help first and who not help first. Pants down, shirts cut off, they had to find the bullets. Just blood everywhere."
Footage from the scene showed dozens of emergency vehicles at the scene and people being treated on the pavements.
Some of the injured were reportedly brought to the Orlando Regional Medical Center in police pick-ups.