Ryanair says the move will benefit passengers in the long-term
Spain's transport ministry has opened an investigation into Ryanair's move to cancel thousands of bookings made via third party internet travel agents.
Ryanair says the sales, which represent about a thousand bookings a day, are illegal.
But the decision, in the height of the summer travel season, has been criticised by consumer groups.
Italy's transport minister called it "another deplorable and incomprehensible move".
Altero Matteoli said the decision not to honour Ryanair tickets booked by third party websites "damages its own clients," in comments published in Italy's daily newspaper La Repubblica.
The no-frills airline announced the move not to accept tickets bought anywhere other than on its website or via its telephone booking line last week.
The news comes as the budget carrier posted a fall of 85% in net profits in the three months to the end of June.
A Ryanair spokesman said all tickets purchased from third parties after 11 August would be invalid and customers would be informed by email and refunded.
"We're cancelling these tickets because they are in breach of our terms and conditions. The third party is breaching our copyright," Daniel De Carvalho told the BBC news website.
The company said that up to 1,000 of the 200,000 bookings made each day on Ryanair flights were carried out via external websites, which it brands "screenscrapers".
He said that customers were sometimes being charged a 200% surcharge on top of the value of the flight when not purchasing the ticket directly.
According to La Repubblica, a European Commission spokesperson said that if the company was found to be cancelling tickets already sold it would intervene.
Reports suggest that tickets bought via sites such as V-tours, of Germany, Spain-based E-Dreams, Ireland's Bravofly as well as some European versions of holiday booking website Opodo could be affected.
Italian consumer associations Adusbef and Federconsumatori have said the move "is in violation of the consumer code and passengers' rights".
E-Dreams has announced that it will take action against the firm for "irresponsibility and negligence", Agence France Presse reports.
But Ryanair defended its choice.
"It's a consumer-friendly decision," Mr De Carvalho said. "We fight for our mission which is to deliver the lowest fares. We believe this measure, in the mid- to long-term, will benefit our passengers."