A recognized television actress, Ashwini Kalsekar pursued her passion for acting soon after graduating with a bachelor`s degree in arts from a college in Mumbai and began training under the guidance of Neena Gupta. She also trained under the well-known theatre acting coach Muzameel Vakil, following which she was actively a part of theatre up until 1995, which is when she started her television career with the critically acclaimed television show Shanti, which featured Mandira Bedi playing the titular role. The very next year, in 1996, she made her movie debut with the Marathi movie Tula Jhapar La.

Post her movie debut, she was only seen being a part of television shows, some of which are Ek Aur Mahabharat, C. I. D., Shaktimaan, and Achanak 37 Saal Baad. However, it has been her negative roles in shows such as Kasamh Se, Bairi Piya, Jhansi Ki Rani, Jodha Akbar, Itna Karo Na Mujhe Pyaar, and Kavach...Kaali Shaktiyon Se that have won her widespread recognition. Meanwhile, the actress returned to the movies in 2003 with The Hero: Love Story of a Spy. Post this, Ashwini has been seen playing supporting roles in movies like Khakee, Aashiq Banaya Aapne, Apaharan, Ankahee, Johnny Gaddar, Golmaal Returns, All The Best: Fun Begins, Golmaal 3, Singham Returns, Badlapur, and Golmaal Again. Her 2018 releases include Andhadhun and Simmba. Her 2019 release includes Wedding Cha Shinema, directed by Saleel Kulkarni. Her 2020 release includes Laxmii. Her 2021 release includes Koi Jaane Na.