Objective: Nowadays, the treatment of infections caused by hospital-acquired resistant bacteria has become very difficult. It is known that hospital infections can be limited and kept to a minimum with the use of appropriate sterilization–disinfection methods. In our study, we aimed to investigate the inhibition effect of ozone—which is low cost, has a nontoxic effect on humans, and does not leave chemical residues and wastes—on resistant microorganisms that cause nosocomial infections. Methods: In this study, 80 strains of bacteria with various resistance patterns and isolated as a causative agent of nosocomial infection were included. Ten strains of each bacterium— MRSA, VRE, MDR Pseudomonas aeruginosa, ESBL (+) Klebsiella pneumoniae, carbapenemase (+) K. pneumoniae, colistin-resistant K. pneumoniae, colistin-resistant Acinetobacter baumannii complex, and colistin sensitive A. baumannii complex—were used. One liter of sterile distilled water (DW) was saturated with ozone for 1 h. A quantity of 0.1 mL of bacterial suspension was added onto 9.9 mL ozonated DW (final bacterial concentration 106 cfu/mL). From the suspensions kept at room temperature, samples were inoculated as a count plate on sheep blood agar with a 10 µL calibrated loop at 10 and 30 min. After 24 h of incubation, the number of growing colonies was calculated by evaluating the Petri dishes. Results: The bacterial inhibition rates of ozonated water at 10 and 30 min exposure times were detected as 97.29–100% for Gram-positive nosocomial-resistant pathogens and 94.76– 99.99% for Gram-negative nosocomial-resistant pathogens, respectively. Conclusion: It has been determined that ozonated water can provide a very high antibacterial effect in vitro at a very low cost. In other studies, the antiviral activity of ozone, including SARS-CoV-2, has also been shown. The data we obtained suggest that ozone can be used in various disinfection–sterilization processes in hospitals. We believe that a cost-effective solution can be produced by supporting such studies with clinical research.