Searching for an alternative disinfection and sanitization strategy to control and prevent the contamination and diseases caused by microbial pathogens represents one of the critical challenges for all world governments. So that the antimicrobial efficiency of ozone gas as a terminal disinfectant was estimated at a relatively small level (1.2 mg/l/h) using a unit that was generated as a local unit assembled at the faculty of science against six reference strains including Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 9372), Bacillus spizizenii (ATCC 6633), Escherichia coli (ATCC 8739), Pseudomonas aeroginosa (ATCC 9027), and Candida albicans (ATCC 10231) for 10, 20, 30 and 40 minutes under laboratory conditions. After 10 min of ozone treatment, the log reduction of cell viability was 97.15%, 59.25%, 24.20%, 24.09%, 14.50 %, 13.47%, and 0.46% for P. aeroginosa, strains combination, E. coli, C. albicans, B. spizizenii, B. subtilis, and S. aureus, respectively. The twenty-minute exposure to ozone resulted in a reduction in microbial viability percent 98.17%, 82.88%, 69.63%, 62.79%, 49.43%, 29.57%, and 28.08%, for P. aeroginosa, strains combination, B. subtilis, and E. coli, C. albicans, S. aureus, and B. spizizenii, respectively. The efficacy of ozone for P. aeroginosa, E. coli, and strains combination increased by more than 98% after 30 min of ozone treatment followed by 90.41%, 86.76%, 52.63%, and 36.64% for B. subtilis, C. albicans, B. spizizenii, and S. aureus, respectively. The maximum ozone efficacy reached 100% for all reference stains except B. spizizenii (62.10%) after 40 min of ozone treatment making this strategy a candidate tool recommended for the management and control of the pathogenic microorganisms.