Sylvie Goulard, a close ally of Macron, had been nominated by incoming European Commission President Ursula von der Leyen for a position overseeing Europe's internal market, industry and defense.
Goulard failed to convince European parliament members, who quizzed her twice about allegations she misused funds and consulted for a U.S. think tank while she served in the EU Parliament.
Goulard is the third candidate to be rejected after lawmakers dismissed a pair of nominees over suspicions of conflict of interest.
She was rejected with 82 votes against, 29 in favor, and one abstention.
Before European lawmakers rejected her nomination, Goulard had denied any wrongdoing in the case dating back to her years in the European parliament from 2009 to 2017. She has not been indicted in a French investigation but resigned as French defense minister when it was opened two years ago.
After an initial hearing left many lawmakers unimpressed and asking for more guarantees, Goulard had to submit written answers to a set of questions before she was quizzed again.
"I gave you all the elements in my possession," she said Thursday. "It's a thrilling mandate. Hopefully you will give me the chance to carry it out."
Lawmakers decided differently, leaving Macron in an uncomfortable position.
Despite her judicial case, Goulard had been hand-picked by Macron, who has portrayed himself as the champion of Europe since he came to power two years ago, pledging to reform the bloc and enhance its sovereignty.
Macron now needs to come up with another candidate that has to be vetted by von der Leyen and approved by lawmakers.
Goulard has been accused of using the funds to pay a legislative assistant who actually worked for her former party. She told lawmakers the issue was related to overpayments she made after the employee stopped working for her and vehemently denied wrongdoing.
In September, she answered questions from French investigators.
"One can never be certain of what will happen, but normally I won't end up being charged," she said.
As for her consultancy work for the Berggruen Institute think tank, she said it was not illegal. Goulard was reportedly paid some 10,000 euros ($11,000) a month for her work with the Berggruen Institute during 2013-2015.
"We obviously did not vote for Goulard," French far-left EU lawmaker Manon Aubry said. "EU citizens won't put their trust in their institutions when a Commissioner sees absolutely no problem in making 10,000 euros a month from a private lobby in addition to her (EU lawmaker) salary."