European Council President Donald Tusk has warned that "worrying declarations" from US President Donald Trump are among the challenges faced by the EU.
He said the change in Washington was part of an external threat that also included an assertive China, an aggressive Russia and radical Islam.
In a letter to 27 European leaders, Mr Tusk also said he believed most of them agreed with him.
Several statements from Washington have prompted alarm in Europe's capitals.
In his letter, issued ahead of an EU summit in Malta this week, Mr Tusk said the new US administration placed the EU in a "difficult situation" as it appeared to "put into question the last 70 years of American foreign policy".
He concluded: "We cannot surrender to those who want to weaken or invalidate the Transatlantic bond, without which global order and peace cannot survive. We should remind our American friends of their own motto: United we stand, divided we fall."
EU unsettled - by Damian Grammaticas, BBC News, Brussels
Donald Tusk doesn't speak for European leaders. His role is to chair their debates and focus their minds on the biggest issues facing the EU.
The way he always does that is by speaking bluntly.
What's striking is that he's added "worrying declarations by the new American administration" to his list of external challenges facing Europe. He does place it lowest, but in Donald Tusk's view the EU is under pressure from all sides.
And now, he's saying, the EU can no longer be sure of the support of its biggest ally too.
Donald Trump has professed doubts about Nato, admiration for Russia's Vladimir Putin and support for Brexit. He has also criticised German Chancellor Angela Merkel, particularly her welcoming policy towards refugees.
So Mr Tusk's letter is a sign of how unsettled EU leaders are by Donald Trump, unsure about what his presidency heralds.
In a recent interview with Germany's Bild newspaper, Mr Trump confirmed his view of the Nato alliance as "obsolete". He has also dismissed the EU as "basically a vehicle for Germany".
Mrs Merkel was among several European leaders who criticised an executive order signed by Mr Trump of Friday establishing a temporary travel ban on seven predominantly Muslim countries.
Mr Trump's statements have raised concerns in Europe, along with his views on trade.
During his inauguration speech, the new president accused foreign competitors of "stealing our companies and destroying our jobs" and said: "Protection will lead to great prosperity and strength."
Last week, Ted Malloch, the man tipped to be named US ambassador to the EU, made less than flattering remarks about the euro when he told the BBC that the currency could collapse in 18 months.
On Tuesday, Belgian Prime Minister Charles Michel tweeted that the US "would be making a mistake if it turned its back on Europe".