Justice Minister Francois Bayrou, leader of the small MoDem party and a key ally of Macron's in the presidential campaign, told AFP: "I have taken a decision not to be part of the next government".
European Affairs Minister Marielle de Sarnez is also set to resign, meaning all three MoDem ministers are leaving the cabinet after just over one month in office.
Defence Minister Sylvie Goulard stood down yesterday, saying she could not carry on while MoDem was facing an investigation into claims it broke European Parliament rules by using funds to pay assistants who are actually based in France.
In exchange, the president rewarded the party for its support by giving them key jobs in his first cabinet -- a caretaker government between the two elections.
France's youngest ever president has pledged to usher in a new era of cleaner politics after a series of scandals involving ministers under his Socialist predecessor Francois Hollande.
Observers say that pledge makes it difficult for Macron to keep MoDem in his new government while it is facing a probe.
Richard Ferrand, a junior minister from Macron's party, has also left the government, this time at the president's request after he became embroiled in a conflict-of-interest scandal involving his wife.
REM took 308 seats in the 577-seat National Assembly, meaning it does not need the support of MoDem, which won 42 seats, to push legislation through parliament.
But Bayrou's departure also deprives Macron of a centrist partner as he seeks to pull together a new government to push forward his pro-business agenda.
Bayrou, 66, was a key backer of Macron's 14-month-old party during the presidential campaign, and his support was crucial in lending legitimacy to the inexperienced candidate.
Government spokesman Christophe Castaner said Bayrou's decision to quit was a "personal choice" which "simplifies the situation".
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