Real Madrid were told to pay back 18.4 million euros ($20.5 million) in one case after the city authorities in the Spanish capital overcompensated them when a land transfer fell through.
The sums were relatively small for clubs that can afford to pay ten of millions of euros in transfer fees for a single player.
"Using taxpayers' money to finance professional football clubs can create unfair competition," Competition Commissioner Margrethe Vestager said in a statement.
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"Professional football is a commercial activity with significant money involved and public money must comply with fair competition rules."
Real Madrid and Spanish champions Barcelona are regarded as the wealthiest clubs in world football with annual turnover of in excess of 500 million euros.
Valencia were ordered to pay back 20.4 million euros as one of three local clubs given cheap loan guarantees by a state-owned credit institution to help them out of financial difficulties.
Hercules must pay 6.1 million euros and Elche 3.7 million on the same grounds, the European Commission said.
Finally, four clubs, Real Madrid, Barcelona, Athletic Bilbao and Atletico Osasuna, benefited from a lower corporate tax rate for more than 20 years, paying 25 percent rather than a 30 percent norm for sports firms. The four would each pay back up to 5 million euros, the Commission estimated.
Separately, the Commission cleared Dutch aid provided to FC Den Bosch, MVV Maastricht, NEC Nijmegen, Willem II Tilburg and PSV Eindhoven, saying this complied with the bloc's rules.
($1 = 0.8987 euros)
(Reporting by Foo Yun Chee; editing by Alastair Macdonald and Keith Weir)
