Seigner was born in Paris, France. She is the granddaughter of actor Louis Seigner (1903-1991). Seigner is the sister of Emmanuelle Seigner and a niece of Françoise Seigner.

She studied comedy at Florent (very famous French school of theater), before making her first appearance in theaters in 1994, alongside her sister Emmanuelle in Smile Claude Miller. She received the Prix Michel Simon in 1995 for her role in Rosine. Two years later, she won a Cesar Award for Best Supporting Actress for her role in Nettoyage à sec.

From 1998 to 2001 she had an affair with the Comedian Laurent Gerra.

She won the "Romy Schneider Prize in 1999. Mathilde Seigner is appreciated for her spontaneity and strong character, she quickly gained favor with the public, thanks to the nostalgic Une hirondelle a fait le printemps (2001) as a city dweller who discovers the joys of the countryside. She appeared in Le passager de l’été, Zone libre, and Dance avec lui. With 3 amis, she returned to comedy, in such films as Tout pour plaire and Détrompez-vous.

Her acting performances have been confined to French-language films, but she has appeared in a number of notable productions. She won Best Actress at the Montreal World Film Festival in 2001 for her role in Betty Fisher and Other Stories and has been nominated for a César Award three times.

From 2002 to 2005, she was in couple with the actor Antoine Duléry.

Since October 2006, Seigner has lived with Mathieu Petit, a cameraman; they have a son.

She returned in 2009 with three dramatic comedies about family (Quelque chose à te dire, Trésor and Une semaine sur deux (et la moitié des vacances scolaires)). After Fini les vacances (2010), she appeared in Camping 2 and the remake of La guerre des boutons (2011).

On 24 February 2012, during the César Award ceremony, she interrupted the telecast when Michel Blanc won, stating she would have preferred that Joey Starr won.