Objective To investigate the sterilization effect of peracetic acid and ethylene oxide on endoscope used for debridement of walled-off pancreatic necrosis (WON) complicated with multidrug-resistant orgamism (MDRO) infection (percutaneous/transgastric). Methods Endoscopes with auxiliary water delivery function used for patients with WON and MDRO infection (percutaneous/transgastric) who underwent debridement in a tertiary first-class hospital in Jiangxi Province were selected and divided into two groups by random number table method: group A was sterilized with peracetic acid immersion, and group B was sterilized with ethylene oxide at low temperature. Specimens of forceps channel, air/water supply channel and auxiliary water supply channel of endoscopes were collected by filter membrane method for microbial culture. The qualified rate of endoscope sterilization, colony number and detection result of MDROs of two groups of endoscopes were compared. Results 78 auxiliary water supply endoscopes were collected, including 39 in group A and 39 in group B, with a total of 234 specimens. The qualified rates of endoscope sterilization of group A and group B were 61.54% and 100% respectively. Qualified rates of forceps channel, air/water supply channel and auxiliary water supply channel in group A were 82.05%, 89.74% and 74.36% respectively, qualified rates of three sites in group B were all 100%. In group A, the total bacterial colonies of forceps channel, air/water supply channel and auxiliary water supply channel were 0-6 CFU, 0-112 CFU and 0-23 CFU respectively. 36 strains of MDROs were isolated, mainly multidrug-resistant Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus and multidrug-resistant Klebsiella pneumoniae. After 15 unqualified endoscopes in group A were sterilized with ethylene oxide at low temperature, qualified rate of microbiological monitoring reached 100%. Conclusion Low temperature sterilization with ethylene oxide is more effective for endoscopic debridement of WON complicated with MDRO infection (percutaneous/transgastric). Microbiological monitoring of endoscope should not be limited to the monitoring of forceps channel, auxiliary water supply channel of endoscope with the auxiliary water supply function should be monitored, so as to reduce the risk of infection.