Biosurfactants are amphiphilic tensioactive natural products that are capable of lowering the surface and interfacial tensions of the growth medium. Efficient biosurfactants are characterized by their ability to enhance the aqueous solubility of hydrophobic compounds and to emulsify hydrocarbons in aqueous medium. Improvement in the fermentation technology, strain selection and use of cheaper and renewable substrates have a vital role in enhancing the production processes of biosurfactant industries. However, large scale production of biosurfactants has not reached a satisfactory economical level due to their low yields. Several studies have reported significant effect of carbon sources on the productivity of biosurfactants by different strains. In the current study medium composition optimization approach was investigated for optimal biosurfactant production using a combination of hydrophobic and hydrophilic carbon sources by Bacillus subtillis CN2 strain, previously isolated from hydrocarbon contaminated soil. The study demonstrated that both quantity and type of carbon sources prompted a significant difference in the amount and activity of the biosurfactant produced. The hydrophobic carbon sources were found to be superior to hydrophilic ones in promoting biosurfactants production and surface activity superiority. The strain produced 10-fold more biosurfactant when growing on oil than when grown on glycerol and significantly higher surface activity as determined from the emulsification index. In addition to using the hydrophobic substrate sunflower oil as a sole substrate, addition of sunflower oil (5%, wt/v) in to the growth medium after depletion of hydrophilic substrate (glycerol) stimulated the production of biosurfactant by more than 200%. Both the type and concentration of the carbon source were shown to be essential determinants of biosurfactant yield and physicochemical properties. The result of our study showed that the presence of optimal hydrophobic substrates in the growth medium triggered release of more biosurfactant through their inductive effect, which shows a promising potential of the approach for large scale viable biosurfactant production.