Nosocomial infections are frequently caused by bacteria that are resistant to various antibiotics, resulting in the mortality or delayed recovery of hospitalized patients. Several studies have investigated the efficiency of ozone (O3) gas for the disinfection of surfaces to eliminate different nosocomial pathogens. In this study, the efficacy of O3 gas in a heavily contaminated healthcare facility was investigated using a low concentration of FDA-approved and human-safe O3. The total microbial loads on the air conditioning (AC) duct, wall, and tables after 1 month of O3 application were 0 CFU/100 cm 2 , 1 CFU/ 100 cm 2 , and 1 CFU/100 cm 2 , respectively. Moreover, the total microbial loads on the AC duct, wall, and tables 2 months after O3 application were 0 CFU/m 2 , 14 CFU/m 2 , and 1 CFU/m 2 , respectively. Finally, after the third month following O3 application, the microbial loads were 0 CFU/100 cm 2 on the AC duct, 7 CFU/100 cm 2 on the walls, and 54 CFU/100 cm 2 on the tables. Overall results show that O3 gas controlled fungal growth, as it was decreased to minimal levels on some swabbed surfaces or even eliminated on most swabbed medical devices and work surfaces. Moreover, O3 is capable of eradicating nosocomial pathogens present in hidden areas even at low concentrations that match the levels approved by the FDA for human exposure. The study concluded that gaseous O3 can serve as an effective, safe, and cheap disinfectant. O3 could effectively work to eliminate both nosocomial bacteria and mould pathogens.