In this study, we have investigated the effects of brine and biosurfactant compositions on crude-oil-rock-brine interactions, interfacial tension, zeta potential, and oil recovery. The results of this study show that reduced brine salinity does not cause significant change in IFT. However, addition of biosurfactants to both high and low salinity brines resulted in IFT reduction. Also, experimental results suggest that the zeta potential of high salinity formation brine-rock interface is positive, but oil-brine interface was found to be negatively charged for all solutions used in the study. When controlled salinity brine (CSB) with low salinity and CSB with biosurfactants were injected, both the oil-brine and rock-brine interfaces become negatively charged resulting in increased water-wetness and, hence, improved oil recovery. Addition of biosurfactants to CSB further increased electric double layer expansion which invariably resulted in increased electrostatic repulsion between rock-brine and oil-brine interfaces, but the corresponding incremental oil recovery was small compared with injection of low salinity brine alone. Moreover, we found that the effective zeta potential of crude oil-brine-rock systems is correlated with IFT. The results of this study are relevant to enhanced oil recovery in which controlled salinity waterflooding can be combined with injection of biosurfactants to improve oil recovery.