Britain launched the historic process of leaving the European Union on Wednesday.
"Those who take aim at European unity threaten their own communities," EU President Donald Tusk told a congress of the European People's Party (EPP) in Malta, which currently holds the presidency of the bloc.
"We must challenge the populists, we must say loud and clear that they are the opposite of modern patriotism.
"Today they are efficiently and cynically taking advantage of social fears and uncertainty, building their own model of security based on prejudice, authoritarianism and organised hatred. Our response must be clear and decisive," he said.
Britain launched the historic process of leaving the European Union on Wednesday, but its European partners were quick to warn of the difficult path that lies ahead amid fears a good deal for London could encourage others to leave.
The centre-right EPP, the biggest party in the European Parliament, approved a document after the two-day congress warning that voters' fears were being exploited by "extremists from both left and right" and "time is running out".
The caution came less than a month before France's presidential election and ahead of Germans going to the polls in September.
"Our open societies, open democracies and open economies within the EU are under threat from those longing for a nostalgic nationalism which would be harmful for our prosperity and which would undermine our values," the EPP said.
Some on Thursday admitted they had underestimated the wave of anti-EU sentiment that saw the British vote with their feet.
"We have not always listened as we should, and we angered people," Irish Prime Minister Enda Kenny told the congress.
Hungary's right-wing Prime Minister Viktor Orban blamed migration, the "Trojan horse of terrorism", for the continent's woes and slammed liberals, saying "leftist politicians are disarming Europe in the face of the Muslim invasion".
The migration issue, the most serious since the end of World War II, has fuelled anger against the EU as it struggles to present a united front on how to tackle the arrival of over one million people fleeing war, poverty and oppression.
German Chancellor Angela Merkel, who opened the door to hundreds of thousands of refugees from Syria and elsewhere in 2015, admitted "we cannot behave as if we do not have an external border".
But she insisted the EU was "a project of peace", with no room for fear-mongers.
And Spanish Prime Minister Mariano Rajoy criticised those who courted voters with false promises, saying "never has a populist party brought its people answers".
The tone was briefly lightened by European Commission President Jean-Claude Juncker, who said Brussels was going to start cutting red tape by getting rid of small, irritating regulations.
"We decided not to annoy people anymore", he quipped, before turning serious and saying that "Brexit is not the end of everything, but we must make it a beginning of something that will be new, stronger and better."
"I suggest we get a move on because otherwise the populists are going to keep running," he said.
Tusk agreed, insisting it was time to stamp out the myth "that we are losing control over our own fates as we delegate part of our powers to the European community... that it is difficult to reconcile being Europeans and patriots".
"This is a view which is both foolish and dangerous," he said.