Objective To explore the disinfection effect of periodic hypochlorous acid infusion on water supply pipelines. Methods Water specimens from 6 water outlets of central sterile supply department were collected at baseline for microbial culture. After introducing a slightly acidic hypochlorous acid water generator, water specimens were collected every 10 days. Continuous disinfection with hypochlorous acid was carried out on the 1st-3rd day, and discontinued on the 4th-10th day. Water specimens of 6 water outlets were collected on the 1st, 4th, 7th, and 10th day of each cycle, in total 3 cycles. Microbial culture and metagenomic next-generation sequencing (mNGS) analysis were conducted simultaneously on all water specimens from the outlets of the endoscope cleaning tank. Results Before disinfection of the pipeline, a total of 18 bottles of water specimens were collected. Bacterial count of water specimens from 10 bottles (55.56%) was between 10² and 10⁴ CFU/mL, mainly Sphingomonas adhesins and Bacillus cereus. After disinfection, only 2 out of 72 water specimens (2.78%) exceeded the bacterial limit. The mNGS results showed that three most dominant bacterial genera in the pre-disinfection water specimens were Sphingomonas, Methylobacterium, and Ralstonia, with stringent mapped reads number (SMRN) ranging from 10 000 to 100 000; while the most dominant bacterial genera in post-disinfection water specimens were Lactobaci-llus, Ralstonia, Acinetobacter, and Bradyrhizobium. PCoA analysis showed that there was a statistically significant difference in β-diversity of water specimens before and after disinfection (P<0.05). Conclusion Periodic hypochlorous acid infusion disinfection can effectively remove bacterial biofilms in water pipelines, but there is a risk of bacterial biofilm recovery during disinfection suspension. In the future, monitoring and management of medical water should be strengthened.