Le Pen, who remains party leader despite calls for her to resign following this year's failed presidential election campaign, has twice denied summonses from judges and police regarding the case.
Under French law, being put under formal investigation means there is "serious or consistent evidence" that points to probable involvement in a crime.
Le Pen's party, the National Front, is already highly critical of the European Union as a whole, so this investigation is not expected to reflect negatively on Le Pen for her base.
Other European parliamentarians, including Le Pen's father Jean-Marie Le Pen and her companion Louis Aliot, have also been on the radar of the European investigative body OLAF for allegedly misusing parliamentary aides' wages.
French authorities charge Marine Le Pen over misuse of European Union funds
Those pictures saw her placed under investigation for the "dissemination of violent images".
She will appeal against the decision on Monday, National Front press director Alain Vizier said.
The French judiciary has already requested that the European Parliament lift Le Pen's immunity. In February, the parliament said it would start to deduct the monies it claimed were owed unless she paid the money back.
The formal investigation of Le Pen comes less than two weeks after she won a seat in the French National Assembly.
The case was first triggered by a complaint about the National Front to the European Parliament.