The purpose of this study was to analyze basic bactericidal and fungicidal activity of ozonated water according to EN 1040 "Chemical disinfectants and antiseptics--Quantitative suspension test for the evaluation of basic bactericidal activity of chemical disinfectants and antiseptics" and EN 1275 "Chemical disinfectants and antiseptics--Quantitative suspension test for the evaluation of basic fungicidal or basic yeasticidal activity of chemical disinfectants and antiseptics" with additional clinical multidrug-resistant bacterial strains and evaluate whether the ozonated water acts as a rapid and efficient antimicrobial agent and as such could be applied during intraoperative ozone treatment for tissue protection against infection with pathogenic bacteria.A prototype device for intraoperative ozone therapy was used. Besides standard bacterial and fungal strains, 60 clinical bacterial isolates were analyzed.The ozone concentration in ozonated water was sufficient to kill almost all cells of the bacterial and yeast strains tested after 30 seconds. Effective action against Aspergillus brasiliensis spores required a longer time than those required in the case of bacterial cells or vegetative cells of yeast.The prototype device used in our study produced high ozone concentrations in freshly prepared ozonated water. This liquid complied with the requirements of the EN Standards: basic bactericidal and basic yeasticidal activities.