Ozone has been one of the most investigated and discussed sanitization methods. This paper reports a procedure to sanitize air hospital environments, in particular chirurgical surgery rooms that require high levels of disinfection. The purpose of this work was the development and implementation of a cleansing and sanitizing procedure for critical clinical settings with ozone, to prevent hospital infections by the elimination of all toxic and harmful microorganisms in the air, and ensure safe use for operators and patients. The protocol for the study involved a structured selection of a representative environment of healthcare structures such as high, medium, and low-risk settings in air and examples of hospital furniture. The concentration of ozone was measured during sanitization treatment and the estimation of the total microbial count in the air and on different surfaces before and after the sanitization operations was performed. The results demonstrated a significant reduction in the microbial count that always fell below the threshold value. Currently, there are no air treatment strategies available for inactivating airborne organisms during hospital outbreaks, which is most probably due to the lack of approved protocols.