US officials refused to directly blame Cuba itself for the "incidents", which appear to have begun last year, and Havana insisted it is working to protect the US mission.
A State Department spokeswoman would not detail the nature or number of the injuries, but she confirmed that a number of US diplomats had returned home for treatment.
Unidentified officials told CNN and other US media that the staff may have been harmed by a sonic device fired either inside or outside their Havana residences.
Some countries have developed sonic and ultrasonic weapons that can be used for crowd control or, for example, to deter seaborne pirates without resource to lethal force.
But there are no known cases of such a device being deployed by hostile intelligence services or terrorists against a diplomatic mission.
State Department spokeswoman Heather Nauert said investigations are ongoing and that Washington is not directly accusing Cuba of being behind the "incidents".
'Major concern'
But - justifying the expulsion of two Cuban diplomats from Washington - she insisted that Cuba, as the US mission's host nation, bears responsibility for its security.
Of the reported victims, she said: "What I can tell you is that these were US government personnel who were in Cuba, in Havana, on official duty on behalf of the US government.
"We consider these to be 'incidents', because we still are trying to determine the actual cause of their situation. They have had a variety of physical symptoms," she said.
"This is an active investigation, and that investigation is ongoing at this time," she said, adding: "It is an area that is under investigation that is a major concern of ours."
Bauert said that US personnel began experiencing ailments in late 2016, but that it was not immediately recognised that it could be anything other than an ordinary health issue.
Cuba's foreign ministry said that US officials had alerted it to "some alleged incidents affecting some officials of that diplomatic mission and their families" on February 17.
Later, after some America staff left, US officials asked two Cuban diplomats to leave Washington in response.
"US government officials have been affected in some way by these incidents -- physically affected by these incidents," she said, explaining the decision.
"It is the Cuban government's obligation under the Vienna Convention to ensure the safety and protection of our diplomats there."
Meanwhile, Canada confirmed that one of its envoys had suffered a similar incident.
"We are aware of unusual symptoms affecting Canadian and US diplomatic personnel and their families in Havana," Canadian foreign ministry spokeswoman Brianne Maxwell said.
Cuba said it had objected to the expulsion of its officials, while also urging the United States to work together to shed light on the incidents in Havana.
"Cuba took this issue with the utmost seriousness and acted expeditiously and professionally in order to clarify the facts of this situation," the foreign ministry said.
The communist state stressed the need for US authorities to share information on the probe and promised to reinforce security around the mission.
Trade embargo
Relations between the United States and Cuba were restored by then US president Barack Obama and his Cuban counterpart Raul Castro in 2015 after a half-century break.
But tensions mounted again alive after the detente was partly rolled back by Obama's successor Donald Trump, who won the votes of many Cuban Americans by promising a tough line.
In June, Trump announced tightened rules for Americans traveling to Cuba, banned ties with a military-run tourism firm and reaffirmed the existing US trade embargo.
The US embassy was closed in 1961 at the height of the Cold War when diplomatic relations broke down between Washington and Fidel Castro's young revolutionary regime.
The mission reopened as a "special interests section" rather than a full embassy under an agreement between Castro and US President Jimmy Carter.
But the US diplomats were hardly made welcome. Anti-American propaganda was displayed around the compound, which Cuba saw as a base for fomenting opposition sedition.
American diplomats in Havana and their Cuban rivals in Washington both regularly complained of harassment or heavy-handed surveillance - but not of sonic attack.
