Ozone attracts great attention due to its strong oxidative properties, antimicrobial activity, easy applicability, operating costs almost negligible, lack of chemicals in its use, highly effective and environmentally friendly application. In this study, two Gram (+) and two Gram (-) bacterial cultures, known as pathogens, were used to examine the effect of ozone gas on the growth of bacterial cultures. The samples were treated with ozone at different flow rates (4, 5, and 6 mg/L) and durations (1, 5, 10, 15, and 20 min) with different application parameters (pathogen bacteria, distilled water, and the mixture of distilled water and pathogen bacteria) and the number of viable cells was determined after the procedure. Among the methods applied we found that the direct application of ozone to the bacteria is the most effective in preventing/destroying bacterial growth. Also, it was determined that the growth of pathogenic microorganisms decreased as the flow rate and ozone contact time enhanced.