Mannosylerythritol lipids (MELs) are novel biosurfactants performing excellent physical-chemical properties as well as bioactivities. This study is aimed to explore the antibacterial and antibiofilm activity of mannosylerythritol lipids against foodborne gram-positive Staphylococcus aureus. The results of growth curve and survival rate revealed the significant inhibitory effect of MELs against S. aureus. The visualized pictures by scanning electron microscope and transmission electron microscope exposed apparent morphological and ultrastructure changes of MEL-treated cells. Furthermore, flow cytometry confirmed that MELs have promoted cell apoptosis and damaged the cell membrane. Notably, MEL-A also exhibited outstanding antibiofilm activity against S. aureus biofilm on different material surfaces including polystyrene, glass, and stainless steel, verified by confocal laser scanning microscope. These findings suggest that the antimicrobial activity of MELs is related to inhibit planktonic cells and biofilm of S. aureus, indicating that it has potential to be an alternative to antibacterial agents and preservatives applied into food processing.