Abstract: OBJECTIVE To analyze the key influencing factors of Pseudomonas aeruginosa biofilm formation in flexible endoscopes in the context of hospital infection risk assessment, and to evaluate the removal effects of different cleaning and disinfection strategies on biofilms, thereby providing experimental basis for the prevention and control of flexible endoscope-associated infections. METHODS Polytetrafluoroethylene (PTFE) tubes were adopted to simulate the lumens of flexible endoscopes. Biofilm models of P. aeruginosa were developed under different temperatures (4, 26, 36 ℃), incubation durations (24, 48, 72 h) and bacterial strains. Crystal violet staining was conducted for quantitative analysis. Mature biofilm models were treated with ultrasonic cleaning, peracetic acid immersion, or a combination of both to compare the efficacy of different cleaning and disinfection strategies. RESULTS Significant differences in biofilm-forming capacity were observed among different P. aeruginosa strains. Environmental temperature markedly influenced biofilm formation, with the highest capacity at 36 ℃, followed by 26 ℃, and the lowest at 4 ℃. Prolonged incubation duration had limited effects on biofilm accumulation. The cleaning and disinfection experiments demonstrated that ultrasonic cleaning combined with peracetic acid immersion reduced biofilm load more effectively than either physical or chemical treatment alone (P<0.05). CONCLUSIONS P. aeruginosa biofilms pose a significant risk for flexible endoscope-associated infections and are influenced by temperature and strain characteristics. This study recommends controlling the storage temperature of flexible endoscopes below 26 ℃ and implementing enhanced cleaning for high-risk endoscopes, providing an experimental basis for infection prevention and control.