The animals, collectively worth more than $US2 million, fell seriously ill as they arrived at the International Polo Club of Palm Beach to play in a fixture as part of the United States Polo Open.
Veterinarians arrived in numbers at the polo grounds to help the horses but the death toll climbed during the Sunday afternoon to 14. A further seven died overnight.
Floirida agriculture commissioner Charles Bronson has ordered an investigation.
Bronson said the rapid onset of sickness and death has led state officials to suspect the deaths resulted from an adverse drug reaction or toxicity.
"At this time there is no evidence that these horses were affected with an infectious or contagious disease as there are no other horses affected at this time."
The dead horses have been taken to a Department of Agriculture laboratory in Kissimmee, Florida, and to the University of Florida Veterinary School for post mortems and toxicology testing.
It could take several days before any test results are in, or a cause of death is known.
"Obviously, this is a tragic situation and we are working hard to determine what happened," Bronson said. "But it would be irresponsible to speculate on what may have killed the horses.
"We will wait until the facts are in before making any specific comments on the case."
The state investigation involves the Agriculture Department's Division of Animal Industry, headed by the state veterinarian, and the department's Office of Agricultural Law Enforcement.
Bronson said agriculture officials will wait until test results are back before determining their next step.
The horses were part of the Venezuela-based Lechuza Caracas team, backed by Venezuelan multimillionaire businessman Victor Vargas, himself a player who reportedly pitched in to help the animals when they became ill.
The team's players are mostly Argentinian.
Its team of horses - which are understood to total about 60 - had been kept at the squad's complex near the polo stadium.
The horses were not showing any signs of illness as of Sunday morning.
When the horses were off-loaded from transporters at the event around 2pm, some of the animals were dead and the remaining horses were showing severe symptoms of depression, respiratory problems, a lack of co-oordination, and an inability to stand.
While some horses died at the scene, others died en route to equine hospitals. The remaining seven succumbed overnight.
Veterinarians have told US media that the deaths appear to have resulted from heart failure triggered by some form of toxin.
The animals were placed on intravenous drips and water was used to try to cool the animals down, without success.
It is understood that every animal that fell ill has since died.
Makeshift tarpaulins were erected around the sick animals at the ground while veterinarians tried to save them.
Meanwhile, the Lechuza Caracas team has withdrawn from the US Open Polo Championship as a result of the deaths.
The US Open Polo Championship dates back 105 years and is one of the most prestigious polo tournaments in the US.