The development of bacterial biofilms in the hydraulic circuit of hemodialysis machines is routinely prevented by frequent use of a variety of chemical and heat disinfection strategies. This study compared the effectiveness of several chemical disinfectants, commonly used either alone or in combination with a treatment regimen that involved cleaning plus heat disinfection using an in vitro Pseudomonas biofilm model. Effectiveness of these procedures was evaluated using total and viable biomass quantitation and polysaccharide and endotoxin determination. The chemical disinfection procedures were only partially successful in removing all biofilm components. Heat disinfection alone killed viable biofilm bacteria, but did not remove all the biomass components, including endotoxin. The combination of cleaning with citric acid followed by heat disinfection was the most effective in eliminating all biofilm components from the hydraulic circuit of the in vitro model.