Along with the two most successful clubs in Spanish football, the Commission has also opened a probe into their fellow top-flight sides Valencia, Athletic Bilbao, Osasuna and Elche.
Segunda Division side Hercules are also under investigation as the Commission seeks to determine if state aid received by those clubs is in line with European Union regulations.
A Commission statement 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."
The Commission said it had concerns that the funding provided significant advantages to those sides to the detriment of other clubs which have to operate without such support.
Explaining what each of the seven clubs is being investigated for, the statement continued: "The Commission will firstly investigate possible tax privileges for Real Madrid, Barcelona, Athletic Bilbao, and Osasuna.
"In another inquiry it will assess whether a widely reported land transfer between the City of Madrid and the club Real Madrid 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, Hercules and Elche, while those clubs were seemingly undergoing financial difficulties."
Joaquin Almunia, the Commission vice-president in charge of competition policy, said: "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."