US Secretary of State Rex Tillerson said on Friday the US mission in the Cuban capital had not been able to determine who was behind what he called "health attacks" that left some staff with hearing loss.
"We hold the Cuban authorities responsible for finding out who is carrying out these health attacks on not just our diplomats but, as you've seen now, there are other cases with other diplomats involved," he said.
Tillerson added: "We hold the Cubans responsible just as every host country has a responsibility for safety and security of diplomats in their country."
His comments came two days after the US said it had expelled two Cuban diplomats after a number of American embassy workers were forced to leave Havana because of mysterious medical symptoms first reported last year.
On Thursday, Canada's government said at least one Canadian diplomat in Cuba also had been treated for hearing loss.
The officials told The Associated Press the hearing loss appeared to have been caused by the deliberate use of some sort of sonic device operating outside the range of audible sound.
Cuba's foreign ministry said US officials had alerted it to the "alleged incidents" on February 17.
The Cuban government said in a lengthy statement on Wednesday that "Cuba has never permitted, nor will permit, that Cuban territory be used for any action against accredited diplomatic officials or their families, with no exception."
Former diplomats and students of US-Cuba relations said they found it inexplicable that Cuba would have tried to harm US and Canadian diplomats, particularly in the fall of 2016 as then president Barack Obama was ending a second term marked partly by the reopening of diplomatic relations with the Caribbean island.
"There'd be no logic to the Cubans trying to deliberately harm US or Canadian diplomats," William LeoGrande, an American University expert on Cuban foreign policy, told AP. "It'd really be unprecedented."
Relations between the US and Cuba were restored in 2015 by Obama and his Cuban counterpart Raul Castro after a half-century break.
But tensions have mounted again after the detente was partly rolled back by Obama's successor Donald Trump.
In June, Trump tightened rules for Americans travelling to Cuba, banned ties with a military-run tourism firm, and reaffirmed the existing US trade embargo.
Source: News agencies