It said Mastercard had prevented retailers using cheaper banking services outside their home country.
Prior to 2015, Mastercard obliged banks receiving card payments to use a fee set in their home country, even if cheaper rates were available elsewhere in the European Union.
Mastercard said the closure of the case was an important milestone.
Commissioner Margrethe Vestager said: "European consumers use payment cards every day, when they buy food or clothes or make purchases online.
"By preventing merchants from shopping around for better conditions offered by banks in other member states, Mastercard's rules artificially raised the costs of card payments, harming consumers and retailers in the EU."
Mastercard is the second biggest supplier of credit card services in the European market.
The fine was reduced by 10% to reflect Mastercard's co-operation with the investigation.
"This decision relates to historic practices only, covers a limited period of time of less than two years and will not require any modification of Mastercard's current business practices," the company said in a statement.