Florida's state attorney general's office said on Thursday that the allegation centres on the Southern Inn in Perry, Florida, a town about 50 miles (80 km) southeast of Tallahassee.
The same district attracted international attention two years ago when a legislator was told in a bar that blacks could only be served in the back room.
Attorney General Charlie Crist issued a subpoena on Thursday to Raj Patel, the Southern Inn's owner, as part of an investigation into possible unfair trade practices and violations of state civil rights laws.
"If the allegations are proven true, these citizens of Florida have been horribly mistreated"
The incident occurred on 18 July, when friends and relatives of Dwayne Parker, who is black, "were repeatedly told by the (motel) owner that 'coloreds are not allowed in the pool,'" a statement from Crist's office said.
"This is a deeply troubling incident," Crist said in the statement. "If the allegations are proven true, these citizens of Florida have been horribly mistreated."
Patel denied the allegations, saying he had asked some of Parker's guests to leave because they were not registered at the motel.
"He had so many kids there.There are rules in the pool that only registered guests are allowed," said Patel, reached by telephone at the motel. "I asked them to leave."
"I didn't say nothing to him about black people can't swim here. If I didn't like black people, why would I rent a room to them?" he said.
Perry also attracted attention two years ago in a case reminiscent of the US South before racial integration.
In that incident, Maryland State Delegate Talmadge Branch said he walked into the Perry Package Store and Lounge on 3 February 2001, to buy a beer and was told to go to the back room were black patrons traditionally drank.
The incident led to civil rights protests in the town. The bar owners were stripped of their liquor license and had to close the business. Branch filed a $30 million lawsuit against them.