Abstract: The quality of cleaning and disinfection of flexible endoscopes is directly associated with the medical safety for their direct contact with patients' mucosal tissues. Biofilm contamination is a key factor for the failed disinfection of the flexible endoscopes and has become a major risk source for health care-associated infections. The biofilm contamination of flexible endoscopes is characterized with universality, site-specificity and high persistence and is affected by multiple factors such as endoscope structure, reprocessing procedures, and storage methods. The research progress in the biofilm formation mechanisms, current status of contamination, clinical issues, monitoring technologies, and disposal strategies was systematically reviewed in the article, with the objective to provide theoretical bases and practical references for infection prevention and control in clinical endoscopy centers and to reduce the incidence of endoscopy-associated infections.