Bacillus sp. strain ISTS2 was applied for the evaluation of biosurfactant production in the presence of various parameters. Initially, various carbon sources were tested in which strain ISTS2 produced biosurfactant in presence of n-hexane (30.16%) followed by CO2 (22.8%) and sodium bicarbonate (18.6%), respectively. These results elucidate that apart from conventional organic carbon sources (n-Hexadecane), inorganic carbon sources like sodium bicarbonate and 5% gaseous CO2 also contribute in biosurfactant production. Utilization of gaseous CO2 in production of value added product make the bacterium more suitable for CO2 sequestration and mitigation of problem of global warming.