Ashwini Kalsekar was born in a Konkani Kokanasth Brahmin family of Goan ancestry on 22 January 1970 in Mumbai, Maharashtra. Her father Anil Kalsekar was a bank employee. Ashwini studied her B.A. from Mumbai and graduated in 1991. She pursued theatre from 1991 to 1994 after her studies. From 1992 to 1993 she was studying under actress Neena Gupta to pursue her dream to be an actress. She is also trained under Muzammeel Vakil, a theatre acting coach from 1992 to 1995. Ashwini is also a trained Kathak dancer.

In 1996, Ashwini played a role in the Marathi film Tula Jhapar La. This was followed by a number of films in Marathi where she appeared in supporting roles.

Ashwini was married to Nitesh Pandey in 1998. They were divorced for unknown reasons in 2002. She married film and television character actor Murali Sharma in 2009.

In 2012, Ashwini was again approached by Ekta Kapoor to play the role of Maham Anga, a foster mother of Emperor Akbar in her historical drama series Jodha Akbar. The role fetched her critical acclaim and major awards. She went on to win the Indian Telly Award for Best Actress in a Negative Role, the Boroplus Gold Award for Best Actress in a Negative Role and other major awards.

In 2012, she was a part of a Marathi comedy reality show, Fu Bai Fu. She was the judge of the show, along with Swapnil Joshi and Sanjay Jadhav, for 2 whole seasons.

In 2014, Ashwini was offered a very different and modern role as Pam Khanna in Ekta Kapoor’s soap opera Itna Karo Na Mujhe Pyaar.