In this work two biological surfactants, a cell-bound biosurfactant produced by Lactobacillus pentosus and a biosurfactant obtained from a fermented stream corn milling industry, were compared with two chemical surfactants (polysorbate 20 and polysorbate 80) in terms of surface tension reduction, critical micellar concentration (CMC), oil spreading and emulsifying capacity. Biological surfactants showed a similar ability to reduce the surface tension of water as polysorbates, which was in conformance with the results obtained in the drop collapse test. Regarding the ability to spread the oil on water, both biosurfactants produced similar results as polysorbates after 1 h. However, after 24 h, polysorbates and biosurfactant from corn stream were more effective than L. pentosus biosurfactant, producing greater free oil areas. Concerning the emulsifying activity, in terms of relative emulsion volume (EV), the biosurfactant produced from L. pentosus gave the best results (EV = 100%), after 1 day of emulsion formation, keeping this value over 50% after 15 days of emulsion formation; whereas polysorbates gave EV values lower than 50%. The CMC values of the biosurfactant from corn stream and of polysorbates were closer in comparison with the CMC value of L. pentosus biosurfactant, observing that the characteristics and properties of the biosurfactant from corn stream are more similar to polysorbates than to L. pentosus biosurfactant. Thus, it could be speculated that biosurfactant from corn stream would be a good substitute for polysorbates