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The present study describes elaborately the isolation of a potential biosurfactant producing and crude oil degrading strains isolated from the reservoir soil of Assam oil reservoir field. The produced biosurfactant was chemically characterized for its applicability for the enhanced oil recovery applications in terms of wetting, interfacial tension (IFT) and oil washing. From the seven isolated strains, Bacillus tequilensis MK 729017 was chosen based on the better surface active properties as it reduced the surface tension to 30±2 mN/m along with a moderate emulsification index of 66±2%. The produced biosurfactant was chemically identified to be lipopeptide, surfactin with a lower critical micelle concentration value of 90 mg/L. The carbon source and environmental parameters were optimized for the maximum concentration of the biosurfactant using response surface methodology based on the central composite design …