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 bisQACs 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.