In a White House press conference on Monday night, Mr Trump took aim at allies, including Australia and Japan, who have declined to directly join the Middle East conflict, which was triggered by US-Israeli strikes on Iran.
"It's not just NATO, you know who else didn't help us? South Korea didn't help us," he said.
"You know who else didn't help us? Australia didn't help us."
Despite Australia providing defensive support by deploying a surveillance aircraft to the Middle East, Australian National University expert associate Jennifer Parker said it was unlikely offensive operations against Iran would be committed.
"I think the Australian government has been very clear that they don't see a role for Australian forces in offensive support," Dr Parker told AAP.
While she does not believe Mr Trump's remarks will fundamentally alter the US-Australia alliance, Dr Parker said it could leave a lasting impression on how Australians viewed the relationship.
"So long after we are through this conflict, I think these comments will be remembered," she said.
"And there will need to be a significant effort to rebuild some of that public trust."
The US president also stepped up his threats to bomb civilian infrastructure in Iran, including bridges and power plants, and brushed aside criticism the action might constitute a war crime.
"The entire country could be taken out in one night," Mr Trump said.
Dr Parker said threats to "take out" Iran should not be read literally, but rather as a political message to Tehran and a signalling of expectations to partners.
"I don't think you can take his statement as literal in any way, shape or form, but it does display frustration," she said.
Asked about his reaction to the threat, Opposition Leader Angus Taylor said Australia's interest from the beginning of the conflict has been to have a free flow of oil.
"It impacts every Australian at the bowser so we want to see the strait opened up," he told Sky News on Tuesday.
Attorney-General Michelle Rowland would not be drawn on Mr Trump's singling out of Australia.
"Australia has taken a position, which is that we are not going to be participating in any offensive way in this conflict," she told reporters in Canberra on Tuesday.
"We will not be deploying troops, but I will reiterate that the Australia-US alliance remains fundamental and we continue to call for a de-escalation of this conflict.
Throughout the conflict, Mr Trump has repeatedly expressed frustration with allies over a lack of support for his proposed naval coalition to help reopen the Strait of Hormuz.
One-fifth of the world's oil supply is transported through the major shipping route, which has been effectively blockaded in response to the war.
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