The importances of bio-surfactants in industrial applications are huge due to their biodegradable and eco-friendly nature. Bio-surfactants mostly find application in cosmetics and health care products. Moreover, bio-surfactants like sophorolipids (SL) also exhibit antimicrobial and skin healing properties. The current studies involve production of SL using low cost substrates like glycerol (15%) instead of glucose (10%) with non-traditional oils (10%) such as jatropha oil, karanja oil and neem oil by using Starmerella bombicola (ATCC 22214). Neem oil gave lower yield i.e. (1.42 g/L) of SL as compared to jatropha oil (4.74 g/L) and karanja oil (5.91 g/L) with glycerol as substrate. Some pretreatment given to crude neem oil like oil refining and ethanol washing with glycerol helps in improved cell growth and SL yield i.e. 2.73 g/L and 3.82 g/L respectively. The comparison thin layer chromatography (TLC), Fourier Transform infrared spectra (FTIR), high performance liquid chromatography (HPLC), liquid chromatography mass spectra (LC-MS) and proton nuclear magnetic resonance spectroscopy ( 1 H NMR) of SL produced on non-traditional oils with glycerol were carried out with that of SL produced on non-traditional oils with glucose. The surfactant properties of sodium lauryl sulphate (SLS) such as surface tension, interfacial tension, stabilization of foam, emulsification, and wetting were improved when SLS was replaced at different concentration of SL.