Prosecutors in Paris opened a probe on Tuesday after a woman filed a complaint alleging that he had abused his powers while mayor of the northern town of Tourcoing in 2015 and 2016.
Darmanin said he had heard about this "new slanderous accusation against me" on the website of Le Point magazine on Wednesday which broke the story of the second complaint against him.
"I don't know the person or what I'm accused of," he told La Voix du Nord newspaper in a report published on Thursday. "I've done nothing wrong... I'm calm, concentrating on my work as a minister and I have confidence in the justice system."
The ambitious 35-year-old right-winger was questioned by police earlier this month over separate allegations that he pressured a former prostitute into sex after she asked for help with a legal problem in 2009.
He denies those allegations too, saying he was a "nobody" at the time, but has admitted to having a reputation as a flirt and to "sending a few persistent SMSs".
The government has closed ranks around Darmanin and popular Environment Minister Nicolas Hulot, who faced allegations last week that he raped the granddaughter of former French president Francois Mitterrand in 1997.
The allegations were never investigated. Hulot, a former celebrity environmentalist, denied them.
The claims have put President Emmanuel Macron in a difficult position given his focus on women's rights which he has promised to make a "national cause."