Abstract Background Treatment of chronically infected wounds is a challenge, and bacterial environmental contamination is a growing issue in infection control. Ozone may have a role in these situations. The objective of this study was to determine whether a low dose of gaseous ozone/oxygen mixture eliminates pathogenic bacteria cultivated in Petri dishes. Methods A pilot study with 6 bacterial strains was made using different concentrations of ozone in an ozone-oxygen mixture to determine a minimally effective dose that completely eliminated bacterial growth. The small and apparently bactericidal gaseous dose of 20 μg/mL ozone/oxygen (1:99) mixture, applied for 5min under atmospheric pressure was selected. In the 2^nd phase, eight bacterial strains with well characterized resistance patterns were evaluated in vitro using agar-blood in adapted Petri dishes (10⁵ bacteria/dish). The cultures were divided into 3 groups: 1- ozone-oxygen gaseous mixture containing 20 μg of O₃/mL for 5 min; 2- 100% oxygen for 5 min; 3- baseline: no gas was used. Results The selected ozone dose was applied to the following eight strains: Escherichia coli, oxacillin-resistant Staphylococcus aureus, oxacillin-susceptible Staphylococcus aureus, vancomycin-resistant Enterococcus faecalis, extended-spectrum beta-lactamase-producing Klebsiella pneumoniae, carbapenem-resistant Acinetobacter baumannii, Acinetobacter baumannii susceptible only to carbapenems, and Pseudomonas aeruginosa susceptible to imipenem and meropenem. All isolates were completely inhibited by the ozone-oxygen mixture while growth occurred in the other 2 groups. Conclusion A single topical application by nebulization of a low ozone dose completely inhibited the growth of all potentially pathogenic bacterial strains with known resistance to antimicrobial agents.