Antitrust officials are also probing the European tax affairs of Apple, as well as the Starbucks cafe chain, putting U.S. multinationals squarely at the center of concerns over low corporate tax deals arranged with small EU states.
President Barack Obama, whose administration is trying to negotiate an ambitious transatlantic free trade deal with the 28-nation bloc, warned earlier this year against Europe turning to protectionism to the detriment of the U.S. tech sector.
Read MoreAmazon embroiled in EU tax crackdown
In the Amazon e-books case, the Commission said it had concerns that the clauses in contracts may make it more difficult for other e-book distributors to compete with Amazon by developing new and innovative products and services.
"Amazon has developed a successful business that offers consumers a comprehensive service, including for e-books," Competition Commissioner Margrethe Vestager said in a statement.
"Our investigation does not call that into question. However, it is my duty to make sure that Amazon's arrangements with publishers are not harmful to consumers, by preventing other e-book distributors from innovating and competing effectively with Amazon."