This study aims to evaluate the cozone bactericidal activity in different suspension media (saline, broth and whole blood) at different exposure times. Methicillin-resistant Staphylococcus aureus, Enterococcus faecalis, ESBL-positive Escherichia coli, MDR Pseudomonas aeruginosa were suspended in different media. We used a bacterial concentration of 0.2 MF for all experiments, as this concentration is consistent with the results of septic shock blood experiments. We performed ozone insufflations in a "sealed environment". The total number of insuffla-tions for each experiment ranged from one to four. The gas concentration was maintained at 80 mcg/ml. We con-firmed the bactericidal activity of ozone on saline for all the bacterial strains. Experiments in broth revealed no changes in the bacterial growth. Ozone is primarily bactericidal against E. coli and bacteriostatic on P. aerugi-nosa, S. aureus and E. faecalis on whole blood. This study confirms the bactericidal efficacy of topical ozone ap-plications and supports the need for further evaluations of the therapeutic potential of major ozone autohemo-therapy. The results in E. coli promote further investigations of ozone activity on other Enterobacteriaceae and its potential use in the treatment of urinary infections. In general, these results suggest that ozone-therapy might be an alternative therapy to overcome antibiotic resistance.