The President is at the centre of a political storm after he signed an executive order restricting entry into the US for nationals from seven Muslim-majority country for 90 days.
Many world leaders have criticised the White House, with many branding the move “unconstitutional”.
Federica Mogherini has issued her own statement condemning Mr Trump, with the 43-year-old Italian pledging continued EU support for Syrian refugees.
She said: “This is not the European Way. The EU will continue to take care and host Syrian refugees and others who are fleeing from war.”
The EU's chief foreign affairs spokesman has criticised Donald Trump
She described the decision to aid Syrians and help construct schools at refugee camps in Lebanon as an “investment” in both their lives and the EU’s own security.
“Every single day a Syrian child is out of school exposes them to a future which is not a future of commitment and engagement,” Ms Mogherini.
In a clear message to President Trump, who has made little secret of his disdain for the EU, Ms Mogherini warned against those who wish to build walls rather than bridges.
She said: “In Europe we have a history that has told that every time one invests in divisions and walls you might end up being in a prison if you build all walls around you.
“We have a history and a tradition that we celebrate when walls are brought down and bridges are built.”
Ms Mogherini vowed the EU would continue to work with the Middle East and criticised Mr Trump for targeting certain religions since he came to office.
We have a history and a tradition that we celebrate when walls are brought down and bridges are built
Her comments followed closely on from Guy Verhofstadt, the European Parliament’s chief Brexit negotiator, who has described Mr Trump a threat to the EU.
Get Quotes on Home Insurance
Speaking in London Mr Verhofstadt said there were three outside forces threatening Brussels.
Along with Vladimir Putin and radical Islam he added: “I have just come back from the US and my view is that we have a third front that is undermining the EU… and that is Donald Trump.”
Ted Malloch, an economics professor who is tipped to be Mr Trump’s ambassador to the EU has hinted he would like to see the EU “tamed”.
Speaking on This Week he said: “The one thing I would do in 2017 is short the euro.
Being President Trump: Key Moments
Fri, January 27, 2017
Inside Donald Trump's first week in the White House.
1 of 12
Trump speaks briefly to reporters as he arrives aboard Air Force One at Joint Base Andrews
“I think it is a currency that is not only in demise, but has a real problem and in fact could collapse in the next year, year-and-a-half.
“I had, in a previous career, a diplomatic post where I help to bring down the Soviet Union. So maybe there’s another union that needs a little taming.”
Mr Trump himself has described the EU as a vehicle for Germany and criticised Barack Obama for warning the UK would fall to the “back of the queue” if it voted Leave.