Kaja Kallas, the EU's foreign policy chief, said the bloc has "no interest to pick a fight, but we will hold our ground".
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European allies have rallied around Greenland's sovereignty. Denmark's foreign minister emphasised the US president cannot threaten his way to ownership of the semi-autonomous Danish territory.
UK Foreign Secretary Yvette Cooper reiterated the UK's position that the future of Greenland is for "Greenlanders and for the Danes alone" to decide.
On Monday, Trump declined to rule out the use of force and insisted he would press ahead with the threatened tariffs on goods arriving in the US from the UK and seven other Nato-allied countries.
Asked by NBC News if he would use force to seize Greenland, Trump answered: "No comment".
The US president said he would charge Britain a 10% tariff "on any and all goods" sent to the US from 1 February, increasing to 25% from 1 June, until a deal is reached for Washington to purchase Greenland from Denmark.
Trump said the same would apply to Denmark, Norway, Sweden, France, Germany, the Netherlands and Finland - all of whom are members of the defence alliance Nato which was founded in 1949.
Asked if he will follow through on the tariff threat, Mr Trump told NBC News: "I will, 100%."
Trump added: "Europe ought to focus on the war with Russia and Ukraine because, frankly, you see what that's gotten them... That's what Europe should focus on - not Greenland."
Denmark has warned that US military action in Greenland would spell the end of Nato. In recent days, Greenland has received support from European members of the alliance - some even sent a handful of troops to Greenland last week in a move seen as symbolic.
However, Trump followed that deployment with an announcement to impose tariffs on the eight Nato allies.
Danish foreign minister Lars Løkke