Cyprus is one of several states, including Israel and Lebanon, racing to tap gas deposits in the eastern Mediterranean.
Greek Cypriots run Cyprus's internationally recognised government, while Turkish Cypriots run a breakaway state recognised only by Ankara in north Cyprus and say resources around the island belong to them too.
Cyprus on Sunday said the Turkish military had obstructed a vessel contracted by Italian oil company Eni which was approaching an area to explore for natural gas.
"Turkey needs to commit unequivocally to neighbourly relations and avoid any kind of source of friction, threat or action directed against a member state," a spokeswoman for the executive European Commission said.
"The EU also stresses the need to respect the sovereignty of member states over their territorial sea and airspace."
Relations between the EU and Turkey have become strained by disagreements over democracy and human rights, especially since a crackdown in the wake of a failed Turkish coup in 2016. However, European countries still rely on Turkey as a NATO ally and to halt the influx of Syrian refugees into the bloc.