In a White House press conference overnight, Mr Trump again took aim at allies who have declined to 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."
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.
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.
"People in Australia and around the world are being impacted by events which they did not cause."
In March, Mr Trump used a blistering social media post to lash a number of countries, including Australia.
He has repeatedly expressed frustration with allies over their lack of support for his proposed naval coalition to help reopen the Strait of Hormuz, which Iran has effectively blockaded in response to the war.
One-fifth of the world's oil supply is transported through the major shipping route.
Nationals leader Matt Canavan backed Labor's decision not to get involved in the conflict, although he said the government should be more clear on what the US had requested of the nation.
"The Australian government is right," he told Sky News.
"We have seen the president make a number of statements in the last few days.
"Some of them have been a little bit weird and wild, but ... I just would like to see the conflict end, because it's having a horrific impact on human lives and the world economy."
