She sang the track Ae Ajnabee with Udit Narayan for A. R. Rahman in Mani Ratnam's Dil Se.. which was her first release as a playback singer and considered to be her debut.

[4] She was part of several successful soundtracks such as Mission Kashmir, Yaadein and Saathiya and worked with some of the biggest music composers like A. R. Rahman, Shankar–Ehsaan–Loy, Vishal–Shekhar, Nadeem–Shravan, Jatin–Lalit and more.

She was known for her performances in songs like "Kabhi Sham Dhale" from Sur – The Melody of Life (2002), "Har Taraf" from Rishtey (2002) and for hit songs like "Chup Chup Ke" from Bunty Aur Babli (2005), "Aaj Ki Raat" from Don: The Chase Begins Again (2006) and "Bol Na Halke Halke" from Jhoom Barabar Jhoom.

Specifically, she sang the Hindi words between the short "Jai Ho" chants, as well as portions of the verses (most of which were sung by Sukhwinder Singh).

In 2013, Mahalakshmi Iyer sang a song with Udit Narayan titled "Ek dil banaaya, phir pyar basaaya", penned by Raghvendra Singh music direction of Navin Manish for Rajshri 's TV show Jhilmil Sitaaron Ka Aangan Hoga on Sahara One.