Background: Patient soil and bacterial biofilm in patient-ready endoscope channels can adversely impact the efficacy of detergent and disinfectant, thereby increasing the risk of nosocomial infection. Biofilm bacteria are firmly attached to one another and to the substrate by exopolysaccharide, making them difficult to remove. We analyzed the effect of 20 wash/contamination cycles on biofilm formation. Materials: Pseudomonas aeruginosa biofilm-covered endoscope tubing was soaked in water (control), an enzymatic cleaner, or a nonenzymatic cleaner (Matrix) for 10 minutes and decontaminated in a washer-disinfector machine. Media containing P aeruginosa was then recycled to simulate contamination in clinical practice. Results: SEM analysis showed that loosely attached biofilm was removed under the high flow rates in the washer-disinfector. The control tubing remained 100% covered with biofilm, which became thicker with increased recontamination cycles. Washing in the enzymatic detergent retarded the redevelopment of biofilm. The nonenzymatic cleaner (Matrix) continued to remove more biofilm with an increasing number of wash/contamination cycles. At the 20th cycle, 90% of the tubing was biofilm-free. Conclusion: Washing endoscopes under high flow rates with some detergents removes established biofilm and retards biofilm generation, emphasizing the importance of cleaning before disinfection. Continued research into the physicochemical mechanisms of biofilm adherence and removal is needed to optimize detergents.