As Lebanon and Israel agreed to a 10-day ceasefire, Mr Trump criticised Australia for not supplying military aid to help reopen the Strait of Hormuz.
"I'm not happy with Australia because they were not there when we asked them to be there," he said.
"They were not there having to do with Hormuz. So I'm not happy. I'm not happy with them."
However, Defence Minister Richard Marles reiterated the US has not made a specific request for military support in the Middle East.
"We'll work with all of our partners, our allies — and that very much includes the United States — in terms of whatever needs to be done in relation to the Strait of Hormuz," he told ABC Radio on Friday.
"I'm not about to go into a running commentary on what the US president says. I mean, we're dealing with the situations as we find them.
"That said, we very much support the strategic objective of denying Iran a deployable nuclear weapon and that capability."
Australia had deployed an E-7A Wedgetail surveillance plane to the Middle East, following request from Gulf countries for defences from missile attacks.
Mr Marles said the federal government was in contact with the Trump administration "at different levels" on a regular basis .
"Our alliance with the United States is as important today as it's ever been, and we continue to work deeply with the US and and that includes communicating to them where we're at," he said.
Opposition defence spokesman James Paterson said the relationship between the US and Australia needed to be prioritised, despite the criticism from the president.
"The reality is he is the president of our most important ally and he will be for the next three years. So this is an important relationship that needs to be managed," he told ABC Radio.
"It is very unfortunate that we have this contradiction between what the Australian government is saying and what the US government is saying."
Senator Paterson said Australia should participate as part of a multinational effort to reopen the Strait of Hormuz.
Treasurer Jim Chalmers, who is in Washington for talks with other finance ministers, said no formal request from the US had been received.
"It's not unusual for President Trump to call for more investment from partners in allies when it comes to defence," he told reporters.
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