This study enrolled 60 patients aged 28 to 76 years who were oral malignancy undergoing radical surgery for more than 3 hours to assess the disinfection effect of povidone-iodine in oral and maxillofacial surgery which is a clean-contaminated wound. The authors collected and compared the sample from oral mucosa and counted the colony-forming units before disinfection, after disinfection for 10 minutes, 1, 2, 3, and 4 hours. The results showed that the oral bacterial colony-forming units significantly decreased after disinfecting with povidone-iodine and the effect existed for 2 hours and the colony-forming units of 3 hours after disinfection showed statistically significant increase. In oral and maxillofacial surgery, povidone-iodine can effectively disinfect the mouth and maintain a certain period of time. Therefore, to reduce the number of oral mucosa microorganisms, it is recommended to disinfect the oral cavity again after 3 hours.