The European Union's diplomatic chief Federica Mogherini said they recognised Turkey faced real security concerns, but echoed US Secretary of State Rex Tillerson's call for Ankara not to go over the top in its response.
Turkey's offensive against the Syrian Kurdish YPG militia has caused alarm in Washington, which views the fighters as important allies in the struggle against IS.
But after talks in Ankara, Tillerson and his Turkish counterpart Mevlut Cavusoglu said they had agreed to work together in Syria, easing tensions.
Mogherini said EU foreign ministers had raised the offensive with Turkish European affairs minister Omer Celik, who joined them at a meeting in the Bulgarian capital Sofia.
"We have expressed as the European Union and member states in recent days and weeks concern about first of all the civilian casualties, about the humanitarian consequences of this military operation," Mogherini said.
"The target of military operations needs to continue to be Daesh (IS) and the UN-listed terrorist organisations. I would subscribe to the words that Rex Tillerson said, that we urge our Turkish friends to show restraint in their military actions."