Infection is the greatest problem in burn patients and topical antimicrobial agents must be chosen with great care, especially when cultured skin is grafted. We examined the cytotoxic effect of six antiseptics and six antibiotics commonly used on cultured human fibroblasts and keratinocytes. Cultured cells were exposed for 15 min to Hibitane (chlorhexidine), Biseptine (chlorhexidine+benzalkonium chloride+benzylic alcohol), Benzalkonium Chloride, Yellow Betadine (polyvidone-iodine+nonoxinol), Betadine Scrub (polyvidone-iodine+quaternary ammonium) and Green Betadine (polyvidone-iodine) and viability was determined using the MTT test. At therapeutic concentrations all the antiseptics are cytotoxic for fibroblasts and keratinocytes. Additionally the cells were exposed for 48 h to vancomycin, colistin, amikacin, imipeneme, pefloxaxin, piperacillin and cell viability was determined using the MTT test. The concentrations of antibiotics corresponding to the plasma peak obtained after therapeutic application were not cytotoxic to the tested cells. The CD50 was much higher than the MIC (from 125 to 875 times for keratinocytes and from 1400 to 5900 times for fibroblasts). These data suggest that commonly applied antiseptics must not be used before grafting cultured skin grafts. After grafting any infection can be controlled with topical applications of appropriate antibiotics