The submarine struck an Iranian frigate with a torpedo near Sri Lanka as part of US attacks, which killed 87 people.
"We wouldn't normally confirm such an issue, but given our (national security committee) meetings and the public interest, I can confirm that there were three Australian persons on board that vessel," Prime Minister Anthony Albanese told Sky News on Friday.
"I can confirm also, though, that no Australian personnel have participated in any offensive action against Iran."
The Australian defence personnel were on board the submarine as part of a training rotation for the AUKUS security pact, which will provide Australia with nuclear vessels.
The prime minister said Australian defence personnel were part of long-standing arrangements to be part of US vessels.
"What they do is ensure that Australian Defence Force personnel, where they're embedded in third countries defence assets," he said.
"They act in accordance with Australian law, with Australian policy, and that, of course, is taking place across the board."
It comes as flights carrying Australians fleeing the Middle East are arriving with empty seats as late-notice airline confirmations and safety concerns keep some citizens from returning home.
Another two flights from Dubai have arrived and several more are scheduled to depart from the war-torn region, as Australians are encouraged to keep their tickets and stay in constant contact with their airlines.
One plane arrived in Sydney on Thursday night and another in Melbourne on Friday morning, bringing home more than 440 Australians.
A flight from Abu Dhabi is expected to land in Sydney on Friday morning.
Emirates is expected to operate another four flights from Dubai in the next 24 hours, while Etihad and Qatar Airways have tentatively scheduled a small number of flights that might not go ahead due to the closure of airspace.
The flights landing in Australia have not always been full as people are only getting a couple of hours' notice from the airlines and some are hesitant to fly because of safety concerns.
The first commercial flight to leave the region for Australia since the outbreak of the US and Israeli conflict with Iran arrived late on Wednesday.
Home Affairs Minister Tony Burke said Australia has been working "really cooperatively" with the United Arab Emirates.
"Our priority is being able to get Australians safe and get them back home," he said.
While the federal opposition has called for repatriation flights to operate in the region for stranded Australians, Mr Burke said regular flights were the best option.
"The simple reality is when you've got more than 100,000 people in the region who have been stranded, a charter flight option isn't going to scratch the surface on that," he said.
"You really need to rely on the commercial airlines."
Opposition foreign affairs spokesman Ted O'Brien said military planes were used at short notice to evacuate Australians from Israel in 2025, New Caledonia in 2024 and Afghanistan in 2021.
There are 24,000 Australians in the UAE, made up of travellers and residents, while about 115,000 are across the broader Middle East.
The federal government has deployed military assets to assist stranded Australian citizens and permanent residents.
A Royal Australian Air Force C17A Globemaster heavy transport aircraft and KC-30A multi-role tanker transport have been deployed as a precautionary measure.
Mr Albanese came under fire from the opposition after he urged Australians to heed travel advice and take up commercial options to return home.
"The government is failing to respond adequately," opposition defence spokesman James Paterson told reporters.
"Every other nation of comparable size and civilians is either chartering aircraft or sending their military planes."
On Thursday, the New Zealand government announced it would send two defence force aircraft to repatriate its citizens.
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