Fellow La Liga sides Valencia, Athletic Bilbao, Osasuna and Elche, as well as Segunda Division outfit Hercules, are also under the spotlight as the Commission attempts to establish whether state aid received by the clubs complies with regulations set by the European Union.
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A statement on the body's website read: "The European Commission has opened three distinct in-depth investigations to verify whether various public support measures in favour of certain Spanish professional football clubs are in line with EU state aid rules.
"None of the measures was notified to the Commission, who was alerted by concerned citizens.
"The Commission has concerns that these measures provided significant advantages to the beneficiary clubs to the detriment of the clubs which have to operate without such support.
"The opening of an in-depth investigation gives Spain and interested third parties an opportunity to comment on the measures under examination; it does not prejudge its outcome."
Going into further detail with regards to the nature of the trio of investigations, the statement continued: "The Commission will firstly investigate possible tax privileges for Real Madrid CF, Barcelona CF, Athletic Club Bilbao, and Club Atletico Osasuna.
"In another inquiry it will assess whether a widely reported land transfer between the City of Madrid and the club Real Madrid CF involved any state aid in favour of the club.
"Finally, it will examine the compliance with EU state aid rules of guarantees given by the State-owned Valencia Institute of Finance for loans that were used to finance the three Valencia clubs Valencia CF, Hercules CF and Elche CF, while those clubs were seemingly undergoing financial difficulties."
Commission vice president in charge of competition policy Joaquin Almunia added: "Professional football clubs should finance their running costs and investments with sound financial management rather than at the expense of the taxpayer.
"Member States and public authorities must comply with EU rules on state aid in this sector as in all economic sectors."