"We can confirm that any tax (or) fees imposed at the border of a member state will be illegal under EU law," Commission spokesman Olivier Bailly told a news briefing in Brussels.
Spain's Foreign Minister Jose Manuel Garcia Margallo had suggested in a media interview at the start of August that a 50-euro (USD 66) tax would be levied for travellers entering Spain from Gibraltar.
The idea provoked outrage amid a growing row around the tiny British outpost at the tip of Spain.
The Commission's reminder of EU law came a day after dozens of Spanish fishing boats sailed to waters around Gibraltar to demand it remove 70 concrete blocks it has dropped in their fishing grounds.
Commission head Jose Manuel Barroso was to speak by telephone with Spanish Prime Minister Mariano Rajoy today, Bailly also said, after the EU leader held a similar conversation with British Prime Minister David Cameron on Friday.
The self-governing British overseas territory of Gibraltar measures just 6.8 square kilometres. It is home to about 30,000 people.
Spain has imposed intense customs checks at the land border, which British Prime Minister David Cameron has described as "politically motivated".
A Commission inspection team is to travel to the border in September to verify that these border checks are legal.
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