"Members of the alliance must finally contribute their fair share and meet their financial obligations," Trump told the heads of state, who stood silently -- some shifting uncomfortably -- behind him.
"Twenty-three of the 28 member nations are still not paying what they should be paying and what they are supposed to be paying for their defense. This is not fair to the people and taxpayers of the United States," he said.
The call appeared to reveal Trump's continued misunderstanding of NATO members' defense commitments. While Trump argued that many of the allies "owe massive amounts of money from past years," the 2% defense spending benchmark that allies must meet is designed to boost their military, not to funnel money to NATO or other allies.
Trump's remarks -- paired with his conspicuous decision not to reiterate US commitment to NATO's mutual defense provision, Article 5 -- are likely to unsettle allies who had hoped to hear Trump assuage their concerns about his commitment to the historic alliance.
Moreover, the President's scolding was cast against a striking backdrop: the freshly unveiled 9/11 memorial, which marks the only time the NATO alliance has invoked Article 5. That decision triggered NATO's participation in the war in Afghanistan.
Remarking on the official opening of NATO's $1 billion new headquarters, Trump offered a tongue-in-cheek comment that he "never asked once what the new NATO headquarters cost."
Trump first cast doubt on the provision during his presidential campaign, shocking members of the alliance when he suggested the US might only defend members of the alliance who "fulfill their obligations to us."
Trump only addressed the Article 5 commitment in passing at the top of his remarks, noting that "our NATO allies responded swiftly and decisively, invoking for the first time in its history the Article 5 collective defense commitments."
He did not promise the US would do the same should a NATO ally come under attack.
Instead, Trump badgered heads of state of the alliance that an increased financial commitment was needed.
Under the NATO treaty, members of the alliance must commit 2% of their GDP to defense spending, a benchmark that only five of the alliance's 28 members currently meet.
"Two percent is the bare minimum for confronting today's very real and very vicious threats," Trump said. "If NATO countries made their full and complete contributions, then NATO would be even stronger than it is today, especially from the threat of terrorism."
Briefing reporters Wednesday on Air Force One, Secretary of State Rex Tillerson said "of course we support Article 5," but declined to say whether Trump would reiterate the US' adherence to the mutual defense pledge in his NATO remarks.
In recent months, Trump has sought to assuage some NATO ally concerns, declaring last month in a joint news conference with NATO Secretary General Jens Stoltenberg the alliance is "no longer obsolete" as he had claimed on the campaign trail.
The President also reiterated his call for stricter controls on immigration and the flow of refugees, saying "the NATO of the future must include a great focus on terrorism and immigration."
"You have thousands and thousands of people pouring into our various countries and spreading throughout. And in many cases, we have no idea who they are," Trump said. "We must be tough. We must be strong. And we must be vigilant."
While Trump signed an executive order to halt the flow of refugees into the US and ban citizens of several Muslim-majority countries, other NATO leaders have opened their countries to refugees and beat back criticism that those policies could increase the terror threat.
Speaking ahead of Trump at the unveiling of a memorial to the fall of the Berlin Wall was German Chancellor Angela Merkel.
NATO allies should remain "united in the trust that it is not isolation and the building of walls that make us successful, but an open society," she said.