Mannosylerythritol lipid-B (MEL-B), which comprises ester-bonded hydrophilic ME and hydrophobic fatty acids, is a bio-surfactant with various unique properties, including antimicrobial activity against most gram-positive bacteria. The gram-positive Staphylococcus aureus is a causative pathogen of dairy cattle mastitis, which results in considerable economic loss in dairy industry. Here, we demonstrate the efficacy of MEL-B as a disinfectant against bovine-derived S. aureus and elucidate a mechanism of action of MEL-B in inhibition of bacterial growth. The growth of bovine mastitis causative S. aureus BM1006 was inhibited when cultured with MEL-B above 10 ppm (equivalent to 0.015 mM). The activity of MEL-B required fatty acids (i.e., caprylic and myristoleic acids) as ME, the component of MEL-B lacking fatty acids, did not inhibit the growth of S. aureus even at high concentrations. Importantly, ME-bound fatty acids effectively inhibited the growth of S. aureus when compared with free fatty acids. Specifically, the concentrations of ME-bound fatty acids and free caprylic and myristoleic acids required to inhibit the growth of S. aureus were 0.015, 10, and 1 mM, respectively. The involvement of ME in the antimicrobial activity of MEL-B was confirmed by digestion of MEL-B with lipase, which dissociated ME and fatty acids. These results indicated that a mechanism of action of MEL-B in inhibiting the growth of S. aureus could be explained by the effective transporting of antimicrobial fatty acids to the bacterial surface via hydrophilic ME.