This study investigates microbial survival in buildup biofilm (BBF) formed in flexible endoscopes during repeated reprocessing cycles. BBF develops due to cyclical exposure to wet and dry phases during use and reprocessing. The research compares BBF to traditional biofilm (TBF), which forms on surfaces constantly bathed in fluid. Using the MBEC system, the study evaluates the effectiveness of glutaraldehyde (GLUT) and accelerated hydrogen peroxide (AHP) in killing microbes in both BBF and TBF. Results show that BBF, formed through repeated cycles of drying, disinfectant exposure, and re-exposure to microbes, provides a more protective environment for microbial survival than TBF. In BBF, the presence of an organic matrix and aldehyde disinfection quickly leads to high levels of organism survival. Cross-linked BBF under high nutrient conditions reached up to 6 Log10 CFU/peg. However, when oxidizing agents like AHP are used and organic levels are low, microbial survival is reduced. The study found that BBF formed by GLUT exposure accumulates microbial load faster than TBF. Post-high-level disinfection (HLD), BBF showed significantly higher survival rates for E. faecalis and P. aeruginosa compared to TBF. Indirect outgrowth testing confirmed microbial survival in BBF. Both TBF and BBF showed surviving organisms when GLUT was used, but AHP survival was less frequent in BBF. The study highlights that BBF is more challenging to disinfect than TBF, especially with GLUT. The data supports the need for thorough cleaning of reprocessed endoscopes, as organic material and microorganisms can prevent effective disinfection. However, cross-linking agents like GLUT are less effective in BBF. The study concludes that BBF allows for faster microbial outgrowth compared to TBF, with significantly higher survival rates when using GLUT. This suggests that the presence of BBF within endoscope channels may reduce the effectiveness of high-level disinfection, increasing the risk of microbial transmission. The findings emphasize the importance of developing improved reprocessing methods to address the challenges posed by BBF in flexible endoscopes.This study investigates microbial survival in buildup biofilm (BBF) formed in flexible endoscopes during repeated reprocessing cycles. BBF develops due to cyclical exposure to wet and dry phases during use and reprocessing. The research compares BBF to traditional biofilm (TBF), which forms on surfaces constantly bathed in fluid. Using the MBEC system, the study evaluates the effectiveness of glutaraldehyde (GLUT) and accelerated hydrogen peroxide (AHP) in killing microbes in both BBF and TBF. Results show that BBF, formed through repeated cycles of drying, disinfectant exposure, and re-exposure to microbes, provides a more protective environment for microbial survival than TBF. In BBF, the presence of an organic matrix and aldehyde disinfection quickly leads to high levels of organism survival. Cross-linked BBF under high nutrient conditions reached up to 6 Log10 CFU/peg. However, when oxidizing agents like AHP are used and organic levels are low, microbial survival is reduced. The study found that BBF formed by GLUT exposure accumulates microbial load faster than TBF. Post-high-level disinfection (HLD), BBF showed significantly higher survival rates for E. faecalis and P. aeruginosa compared to TBF. Indirect outgrowth testing confirmed microbial survival in BBF. Both TBF and BBF showed surviving organisms when GLUT was used, but AHP survival was less frequent in BBF. The study highlights that BBF is more challenging to disinfect than TBF, especially with GLUT. The data supports the need for thorough cleaning of reprocessed endoscopes, as organic material and microorganisms can prevent effective disinfection. However, cross-linking agents like GLUT are less effective in BBF. The study concludes that BBF allows for faster microbial outgrowth compared to TBF, with significantly higher survival rates when using GLUT. This suggests that the presence of BBF within endoscope channels may reduce the effectiveness of high-level disinfection, increasing the risk of microbial transmission. The findings emphasize the importance of developing improved reprocessing methods to address the challenges posed by BBF in flexible endoscopes.