On arriving in the Turkish capital on Tuesday, Trump took swipes at allies for not standing by the US on the Iran war and said he might have boycotted the meeting had it not been for his friendship with the host, President Recep Tayyip Erdogan.
The US then unleashed new military strikes on Iran and revoked a licence allowing Iran to sell oil in response to attacks on three tankers.
It was the latest blow to a fragile ceasefire agreement in a war that is deeply unpopular in Europe.
NATO Secretary-General Mark Rutte defended the new US strikes as "absolutely necessary" and played down Trump's disappointment with allies as "isolated cases".
NATO on Tuesday had sought to demonstrate that its European members were heeding Trump's calls to spend more on their own defence and rely less on the US by unveiling a raft of arms deals worth at least $US50 billion ($A72 billion).
Trump, who has harshly criticised NATO during both his first and second terms in office, said he was "very disappointed" with the alliance and that the US was not "treated well" during the US-Israeli war on Iran.
"Why are we spending hundreds of billions of dollars, and they're not there for us? We've always been there for them," Trump said in an appearance on Tuesday alongside Erdogan.
Trump has accused European nations of failing to let US forces use their airspace and bases on their territories during the war.
European officials have said they largely honoured their commitments to US forces, despite not having been consulted about a conflict that roiled their economies.
In recent weeks, Trump has singled out Italian Prime Minister Giorgia Meloni - formerly a close ally - for criticism.
On Tuesday, Trump said his relationship with Meloni "became a little bad because she refused to help us" with Iran while also describing her as a "nice person".
Trump also said Greenland, a semi-autonomous territory of NATO member Denmark, should be controlled by the United States.
Such assertions have long strained relations between Washington and Copenhagen - both founding NATO members - and more broadly US ties with Europe.
"We are ready to defend every inch of NATO, including our own territory ... Of course we will defend the kingdom of Denmark," Danish Prime Minister Mette Frederiksen said in Ankara, reiterating that Greenland was not for sale.
"One of the reasons why we have built NATO many, many years ago, is if anything happens to one of us, then everybody should stand up for each other," she said.
The 32 NATO leaders met for a dinner on Tuesday evening but the main session of their summit takes place on Wednesday.
Ambassadors from all NATO members have approved a summit declaration that affirms an "ironclad commitment" to collective defence.
But the declaration will only be issued after it has been endorsed by the leaders.
The Trump administration has pressed Europeans to boost defence spending and take on primary responsibility for the conventional defence of Europe as it seeks to shift its military focus to the Indo-Pacific.
The US has also announced troop withdrawals from Europe, cut the forces it assigns to NATO's defence plans - including an aircraft carrier, refuelling aircraft, fighter jets and drones - and launched a six-month review of its military presence on the continent.
European leaders say they are working to take on more responsibility for the continent's security but want a predictable and orderly transition to avoid any gaps in their defences that could be exploited by Russia.
