The bactericidal activity of commonly used antiseptics against clinical isolates was determined. It is noteworthy that povidone-iodine (PVP-I) solution showed high bactericidal activity against all of the test strains after 30 s of exposure. However, in the case of chlorhexidine gluconate (CHG), residual bacteria were observed in most species. Next, acquisition of resistance to antiseptics was examined, and it was found that remarkable increases in MICs were seen for CHG and alkyldiaminoethylglycine hydrochloride. The strains which acquired resistance against one antiseptic showed cross-resistance to all antiseptics except for PVP-I. As for bactericidal activity against biofilm, no viable cells were seen after a 10-min exposure to PVP-I solution. No decrease in viable cell count was seen even after a 60-min exposure to any of the other antiseptics. PVP-I showed high activities in all the tests conducted in this study. Thus, PVP-I was confirmed to be a clinically useful antiseptic.