Background: Biofilm consisting of bacteria enclosed in a matrix of exopolysaccharide (EPS) forms on many medical devices such as catheters and implants. Nosocomial infection is, thus, a newly recognized scenario of biofilm development. Biofilm removal by physical methods such as ultrasound and mechanical cleaning is reasonably effective but difficult to supervise in practice. Chemical methods are often ineffective because of biofilm resistance to biocides. In this study, we compared the efficiency of different detergents used in endoscope reprocessing. Methods: Escherichia coli biofilm was generated on Teflon and medical grade PVC tubing under low flow conditions. Sections of biofilm covered tubing were washed using test detergents and biofilm removal was assessed by counting remaining adherent bacteria after washing and by scanning electron microscopy to qualitatively assess the amount and nature of the remaining biofilm. Results: Control tubing developed a multilayered biofilm consisting of > 105 bacterial cell s/cm2. Only Matrix (Whiteley Medical, Sydney, Australia) produced > 4 log reduction in viable bacterial numbers. Matrix and Epizyme Rapid (3M Australia, Pymble, Australia) were able to remove up to 75% and 60% of the biofilm, respectively. Conclusions: Many commonly used enzymatic cleaners fail to reduce the viable bacterial load or remove the bacterial EPS. Cleaners with high enzyme activity, Epizyme Rapid, removed more biofilm but failed to reduce bacterial numbers more than 2 logs. The only cleaner containing no enzymes, Matrix, significantly reduced bacterial viability and residual bacterial EPS.