Background: Patient-ready endoscopes were monitored over an 80-week period to determine the efficacy of decontamination procedures in a busy endoscopy center. Decontamination failure was related to patient and procedural parameters. Methods: Samples from patient-ready endoscopes were cultured aerobically and anaerobically and subjected to polymerase chain reaction (PCR) to detect hepatitis B virus (HBV), hepatitis C virus (HCV), and HIV. PCR to detect coliforms from 109 culture negative washes was used as a surrogate marker for biofilm in endoscopes. PCR was used to detect the presence of Helicobactor pylori in endoscopes used on infected patients. Procedural information such as biopsy retrieval, endoscope number, diagnosis, attending personnel, and decontamination system procedures was collected. Results: Gastroscopes (n = 1376) and colonoscopes (n = 987) were equally contaminated (1.8% vs 1.9%, respectively) with low numbers of organisms commonly isolated from the nasopharynx and/or feces. Only 1 wash contained viral nucleic acid (HCV). There was a significant correlation (P < .001) between the number of times a patient-ready endoscope was contaminated and its frequency of use. Colonoscopes used on patients with gastrointestinal disease were significantly more likely to remain contaminated through the decontamination process (P < .05). All other patient, staff, and decontamination system parameters remained not statistically significant. Coliform DNA was detected in 40% of culture-negative washes collected from patient-ready endoscopes, suggesting the presence of biofilm. No H pylori DNA was detected. Conclusion: Recommended decontamination procedures do not entirely eliminate persistence of low numbers of organisms on a few endoscopes, but this is unlikely to cause serious consequences in patients. Bacterial biofilm is difficult to remove and may explain this low-level persistence.