Ensuring air quality in operating rooms is crucial for the health and safety of healthcare professionals and patients. This study, focused on 141 operating theatres in the Campania Region from 2015 to 2022, highlights the importance of air monitoring in operating rooms. Microclimatic parameters, air exchanges, thermal comfort indices, air pressure differences, and anesthetic gas concentrations were measured using standardized procedures. Results indicate that 19% of microclimatic checks exceeded acceptable limits, with significant non-compliance in air velocity (0.01–0.04 m/s, mean 0.03 m/s) and air changes (1–14 h−1, mean 6 h−1). Additionally, levels of anesthetic gases such as nitrous oxide (54.7–197.31 ppm, mean 142.92 ppm) and sevoflurane (2.05–19.45 ppm, mean 5.90 ppm) frequently exceeded recommended exposure limits, raising health concerns. These findings underscore the importance of rigorously adhering to environmental standards and continuously monitoring for optimal conditions in operating rooms. The study also stresses the necessity of ongoing education and training for healthcare personnel on preventive measures to reduce risks. In conclusion, maintaining optimal environmental conditions not only safeguards the comfort and safety of healthcare professionals and patients, but also improves team productivity and clinical outcomes. The study advocates for regularly updating national guidelines and rigorously enforcing safety protocols in healthcare facilities.