The antimicrobial properties of the skin are attributed to several agents including human beta-defensins (hBDs), cathelicidin LL-37 and skin lysozyme. Although these antibacterial agents reside in the skin to protect it against infection, it is not well known whether the total analysis of all combinations of these agents may result in synergistic effect to enhance their antibacterial activities against invading microorganisms. To elucidate the interactions between keratinocyte-derived antibacterial agents in the extracellular milieu, we investigated the individual and synergistic activities of hBDs, LL-37 and lysozyme against Staphylococcus aureus and Escherichia coli in neutral and acidic milieus. The colorimetric method using alamarBlue was employed to assess the antibacterial activities of hBD-1, -2, -3, LL-37 and lysozyme and the viability of bacteria was read spectrophotometrically. In both neutral and acidic pH milieus, hBD-1, -2, -3, LL-37 and lysozyme exhibited antibacterial activity against S. aureus and E. coli in a dose-dependent manner. Interestingly, the antibacterial activity of hBD-1, -2, -3 and lysozyme but not LL-37 was significantly enhanced in acidic milieu (pH 4.6). Furthermore, various combinations of above agents resulted in a synergistic or additive antibacterial effect against S. aureus and E. coli in neutral milieu. The synergistic effect of hBDs, LL-37 and lysozyme against S. aureus was further significantly enhanced in acidic milieu. In contrast, above antibacterial agents exhibited mainly additive rather than synergistic effect on antibacterial activity against E. coli in acidic milieu. Taken together, these results provide a novel evidence of antimicrobial mechanism of natural human skin-derived antibacterial agents against bacterial infection, and their involvement in innate immunity.