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Background: With increasing bacterial antibiotic resistance and an increased infection risk due to more complicated surgical procedures and patient populations, prevention of surgical infection is of paramount importance. Intraoperative irrigation with an antiseptic solution could provide an effective way to reduce postoperative infection rates. Although numerous studies have been conducted on the bactericidal or cytotoxic characteristics of antiseptics, the combination of these characteristics for intraoperative application has not been addressed. Methods: Bacteria ( Staphylococcus aureus and S. epidermidis ) and human cells were exposed to polyhexanide, hydrogen peroxide, octenidine dihydrochloride, povidone-iodine, and chlorhexidine digluconate at various dilutions for two minutes. Bactericidal properties were calculated by means of the quantitative suspension method. The cytotoxic effect on human fibroblasts and mesenchymal stromal cells was determined by a WST-1 metabolic activity assay. Results: All of the antiseptics except for polyhexanide were bactericidal and cytotoxic at the commercially available concentrations. When diluted, only povidone-iodine was bactericidal at a concentration at which some cell viability remained. The other antiseptics tested showed no cellular survival at the minimal bactericidal concentration. Conclusions: Povidone-iodine diluted to a concentration of 1.3 g/L could be the optimal antiseptic for intraoperative irrigation. This should be established by future clinical studies.