Josep Borrell, the EU's high representative for foreign affairs, said Washington's secondary sanctions on those doing business with its foes were hitting European companies carrying out "legitimate business."
He also pointed to sanctions on Iran, Cuba and the International Criminal Court, saying the EU opposed the US policy of enforcing its measures against companies and individuals around the world.
"I am deeply concerned at the growing use of sanctions, or the threat of sanctions, by the United States against European companies and interests," Borrell said in a statement.
"Where common foreign and security policy goals are shared, there is great value in the coordination of targeted sanctions with partners.
"Where policy differences exist, the European Union is always open to dialogue. But this cannot take place against the threat of sanctions."
Borrell's statement comes just two days after President Donald Trump's administration moved to include Nord Stream 2 in tough measures that could include severing access to the US financial system.
Germany is a major player in the pipeline project, which will nearly double Russian gas shipments to Europe's largest economy.
The US move means German companies will suffer sanctions for even small investments.