Applegate married actor Johnathon Schaech on October 20, 2001. Their relationship was highly publicized, and they were often seen together at Hollywood events.

Applegate and Schaech divorced in August 2005. The reasons for their separation were not publicly disclosed in detail, but it was reported as an amicable split.

Applegate was diagnosed with breast cancer in 2008. She underwent a double mastectomy and reconstruction. Her openness about her experience made her a prominent advocate for breast cancer awareness and early detection.

Following her breast cancer diagnosis, Applegate founded Right Action for Women, a charitable organization dedicated to providing education and screening resources to women at increased risk of breast cancer.

Applegate married musician Martyn LeNoble on February 23, 2013, after several years of dating. LeNoble is a founding member of the alternative rock band Porno for Pyros.

Applegate and LeNoble welcomed their daughter, Sadie Grace LeNoble, on January 27, 2011. Motherhood became an important part of Applegate's life, and she often spoke about the joys and challenges of raising her daughter.

Applegate publicly announced her diagnosis of multiple sclerosis (MS) in 2021. She shared her challenges with mobility and pain, and how MS impacted her life. She has become an advocate for MS awareness and research.