Objective To explore the optimal morning discharge time and pipeline disinfection frequency of the final rinse water from the endoscopy center according to the microbial culture results. Methods Different morning discharge timing and number of bacterial colonies in the pipeline for the final rinse water from the endoscopy center of a hospital in Jiangsu Province were monitored. Microbial detection was conducted on water samples collected from the two final rinse water sampling sites in the endoscopy room after 0, 1, 3, 5, and 7 minutes of discharge, respectively (100 mL each, continuously monitored for 35 days, with 70 samples at each time point) to explore the optimal discharge timing. The optimal disinfection frequency of pipelines for purified water was explored according to the determined optimal morning discharge timing. Two samples were taken daily from 2 sampling sites after pipeline disinfection and continued for 5 weeks, resulting in 70 samples in total. Results Sampling and monitoring of the final rinse water at different morning discharge time points showed that the median numbers of bacterial colonies at 0, 1, 3, 5, and 7 minutes were 745.00 (373.00, 1 452.50), 150.00 (96.75, 235.75), 44.00 (38.00, 48.00), 12.00 (5.00, 18.00), and 6.00 (4.00, 9.00) CFU/100 mL, respectively. Except the difference between the 5 minute group and the 7 minute group (P>0.05), differences among all the other groups were statistically significant (all P < 0.05). The median of the 0, 1, 3, and 5 minute groups was > 10 CFU/100 mL, while the median of the 7 minute group was < 10 CFU/100 mL, within the qualified range. The discharge time was therefore determined to be 7 minutes. The average numbers of bacterial colonies from the final rinse water samples taken at different time points after pipeline disinfection (1, 2, 3, 4, and 5 weeks after disinfection) were (4.21±0.86), (4.43±0.71), (6.00±0.56), (6.43±0.45), and (13.57±1.03) CFU/100 mL, respectively. The qualification rates of pipeline in terms of bacterial colony were 100%, 100%, 100%, 100%, and 28.57%, respectively. The differences of average numbers of bacterial colonies from the final rinse water samples taken at different time points after pipeline disinfection were statistically significant (P < 0.001). The average number of bacterial colonies 5 weeks after pipeline disinfection was different from those after 1, 2, 3, and 4 weeks, with statistically significant differences(all P < 0.05), while no statistically significant differences among the other groups were observed(all P>0.05). The optimal disinfection frequency for the purified water pipeline was once every 4 weeks. Conclusion The final rinse water remaining in the terminal of pipeline is contaminated to a certain degree. It is recommended to discharge water in the morning for 7 minutes before using it, and disinfect the purified water pipeline every 4 weeks.