In this study, a biosurfactant produced by a Bacillus subtilis strain isolated from oil-contaminated soil from an Omani oil field was tested for its potential in enhancing oil recovery by a series of coreflooding experiments. It was found that the performance of the biosurfactant was increased by mixing with chemical surfactants, by which the maximum production went up to 50% of residual oil at a mixing ratio of (50:50). The second objective of this study was to investigate the effects of the biosurfactant on wettability alteration and to estimate its tendency to loss caused by adsorption. The influence of biosurfactant on wettability was studied by contact-angle measurements, atomic force microscopy (AFM) technique on few-layer graphene (FLG) surfaces, and Amott wettability tests on Berea sandstone cores. Contact-angle measurements showed that the wettability of the biosurfactant solution changes to more oil-wet as the angle decreased from 70.6 to 25.32° when treated with 0.25% (w/v) biosurfactant solution. Amott testing showed a change in wettability index from strongly water-wet in the untreated core toward less water-wet in biosurfactant-treated cores. These results confirmed the ability of the biosurfactant to alter the wetting conditions against different surfaces, thereby serving as a mechanism for enhancing oil recovery. The maximum loss of biosurfactant caused by adsorption was 1.2 mg/g of rock, which is comparable with reported chemical-surfactant values.