Many studies have investigated the effects of pH, temperature, and salinity on the surface-active properties of various surfactants, although in most cases the variables have been studied separately, without considering the effects of any interactions between them. In the present study, a Box-Behnken factorial design was applied to study the effects of pH, temperature, and salinity on the surface-active properties of a biosurfactant produced by Lactobacillus pentosus. The data obtained enabled development of a second-order model describing the interrelationships between operational and experimental variables, by equations including linear, interaction, and quadratic terms. The variable that had the greatest effect on the surface-active properties of the biosurfactant was pH. Moreover, at pH 3-5.5, decreases in salinity and temperature acted synergistically, reducing the surface tension of the biosurfactant; at pH 8, the same effect was observed with increasing salinity and temperature.