Brussels has opened two separate investigations, the first into proposed additional cooperation between British Airways, American Airlines and Iberia.
The second probe is into existing and planned cooperation between Air Canada, Continental, Lufthansa and United.
The commission says it suspects they have colluded too closely on ticket prices and route schedules.
BA said the commission had stressed it did not have proof of any infringement.
The investigations are into transatlantic routes between Europe and North America run by One World alliance members BA, American and Iberia, and Star Alliance carriers Air Canada, Lufthansa and United.
Continental is not yet a member of the Star Alliance but is in talks to join the group.
The commission said there was no deadline for the completion of the investigation.
The BBC reports the investigation comes a year after an "Open Skies" agreement between the European Union and the United States. The agreement aims to enable more airlines to start transatlantic routes.