In food processing, both preservatives and surfactants are usually added to ensure the quality of food. Sophorolipids (SLs) are extracellular biosurfactants produced by yeasts that have antibacterial activities. Nisin is a polycyclic antibacterial peptide produced by Lactococcus lactis. In this study, the possibility of using SLs alone as a preservative and in combination with nisin as a compound additive is investigated. SLs and nisin exhibit effective antibacterial activities against foodborne pathogen Staphylococcus aureus with minimum inhibitory concentrations of 32 and 0.5 µg mL−1, respectively. Both SLs and nisin increase cell wall and cell membrane permeability of S. aureus, enhance intracellular content release and activities of extracellular ALP and β‐galactosidase, and reduce respiratory‐chain dehydrogenase activity of the pathogen. Fractional inhibitory concentration (FIC) index for SLs and nisin combination is 1.5, indicating the effect of SLs with nisin is additive. Results of the study indicate that SLs could be used alone as a preservative or in combination with nisin as a promising compound additive with functions of both emulsifier and preservative. Practical Applications: SLs could be used alone as a preservative in food industry or in combination with nisin as a promising compound additive with functions of both emulsifier and preservative.