The necessity of using ozonated oil and water for treating infections such as gonorrhea and meningitis which their treatment by common antibiotics is sometimes difficult and it is becoming more apparent every day. In this study, the antibacterial effect of ozonated oil and water against Neisseria gonorrhoeae (N. gonorrhoeae) and N. meningitidis were investigated using Broth microdilution methods at zero, 24 and 48 hours after incubation at 37°C. The results were determined by culturing bacteria on their specific culture medium and using an ELISA reader to determine minimum inhibitory and bactericidal concentrations (MIC and MBC, respectively). The results showed that ozonated oil with PI=500 had an expressive effect on N. meningitidis. The concentration of 0.09PI was determined as MIC and the concentration of 0.19PI was determined as MBC. The ozonated oil with PI=1000 had an expressive effect on N. gonorrhoeae. The concentration of 3.12 PI was determined as MIC and the concentration of 6.25 PI was determined as MBC. Ozonated water containing 2.5 mg/L of ozone had no significant antimicrobial effect on the studied bacterial species. The results of this study showed that ozonation of oil may improve its chemical properties. In addition, by increasing the incubation time to 24 hours, ozonated oil showed a favorable antibacterial effect against N. gonorrhoeae and N. meningitidis.