With the object of developing new biocides milder for human use than the current antiseptics, we synthesized a series of bis-quaternary ammonium compounds (bis-QACs). The antimicrobial activity of the newly synthesized bis-QACs and common biocides used as antiseptics was compared by examining minimum inhibitory concentrations and minimum bactericidal concentrations (MBCs). Moreover, the cytotoxicity of these compounds against human cells was determined to calculate the biocompatibility index (BI) of these compounds. BI was the ratio of the concentration of a biocide giving a 50% lethal effect on normal human epidermal keratinocytes to its MBC against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. The commonly used antiseptics tested were benzalkonium chloride (BAC), octenidine dihydrochloride (OCT), chlorhexidine digluconate (CHG) and polyhexamethylene biguanide (PHMB). In comparison with these antiseptics, it was shown that some of new bis-QACs exhibited a wider and more potent antimicrobial spectrum than OCT. The cytotoxicity of these bis-QACs was equal or lower compared to that of the quaternary ammonium compounds (BAC and OCT), although these bis-QACs showed higher toxicity than the biguanide-based compounds (CHG and PHMB). Finally, the comparison of BIs revealed that new bis-QACs such as N-dodecyl ｛4,4'-(2,4,8,10-tetraoxaspiro［5.5］undecan-3,9-diyl) ｝dipyridinium dibromide (4TOSU-12), 3,3'-［1,4-Phenylenebis (oxy)］bis (1-dodecylpyridinium) dibromide (3PHBO-12) and 3-(3-Hydroxy-2-(hydroxymethyl)-2-｛［(1-dodecylpyridinium-3-yl) oxy］methyl｝propoxy)-1-dodecylpyridinium dibromide (3HHDMP-12) had equal or greater biocompatibility than the commonly used biocides tested. Thus, these results strongly suggested that 4TOSU-12, 3PHBO-12 and 3HHDMP-12 could be useful as antiseptics for topical application to the skin.