Abstract: Objective: To evaluate the effect of standardized cleaning and disinfection procedures without self-cleaning time in sequential Operating Rooms (ORs), and to provide a reference for controlling hospital infections and improving operational efficiency. Methods: A total of 537 sequential operations performed in clean ORs were enrolled from December 2025 to January 2026 across 11 tertiary hospitals in Beijing, Shanghai and Zhejiang by convenience sampling method. Through unified training and assessment, implement a standardized cleaning and disinfection procedures with no self-cleaning time. On-site observations documented operation time and step completion, while environmental surface sampling was performed according to "hygienic standard for disinfection in hospitals". Healthcare worker satisfaction was surveyed. Results: After implementing standardized cleaning and disinfection procedures without self-cleaning time, the overall compliance rate for total bacterial colony counts on environmental surfaces was 95.44%. The complete execution rate for key cleaning and disinfection steps reached 97.30%. The median duration of cleaning and disinfection was 4.80 minutes. Class 1,000 ORs demonstrated the shortest cleaning time (3.00 minutes) and highest compliance rate (96.18%). The median turnover time was 14.30 minutes. The overall satisfaction rate among surgical team members was 95.23%. Conclusion: The standardized cleaning and disinfection procedures without self-cleaning time can effectively control environmental microbial contamination and demonstrates excellent applicability across different air purification levels in ORs. This protocol successfully balances hospital infection control with operational efficiency and offers a replicable solution for ORs management optimization, worthy of popularization.

Key words: sequential operation; operating room; cleaning and disinfection; multicenter study; control of nosocomial infection; operational efficiency