Oil recovery potential of surfactant flooding is restricted due to the inability of single surfactant to achieve ultra-low interfacial tension (IFT) and strong water-wet conditions. To overcome these limitations, surfactant mixtures are employed to achieve the desired enhanced oil recovery (EOR) properties. In this study, the efficacy of a combined bio and synthetic surfactants system as a potential means of enhancing oil recovery was examined. The surface, interfacial, and wettability alteration properties of biosurfactant, synthetic surfactant, glycerol, and their combined mixture were investigated. The results revealed that the combined surfactant system had a more pronounced effect on oil-water IFT and wettability alteration compared to the individual solutions separately. Ultralow IFT (0.005 mN/m) was obtainable using the combined surfactant system. The core flooding experiments conducted on Gray Berea sandstone cores using Assam crude oil showed that an additional 15–20% of original oil in place (OOIP) could be recovered with the combined surfactant system. Additionally, simulation studies showed that on a field-scale incremental oil recovery of 12–15% OOIP could achieve depending on the injection mode. Thus, the study revealed that through the unique combination of bio and synthetic surfactants with glycerol, a competent EOR fluid could be developed.