AimsThis work aimed to assess at pilot scale the effect of chlorination and water flushing on 2-month-old drinking water biofilms and, above all, on biofilm-associated F-specific RNA bacteriophages MS2, GA and Q. Methods and ResultsChlorination (4mgl(-1)) was applied first with a hydrodynamic shear stress of 1Pa and second with an increase in hydrodynamic shear stress to 10Pa. Despite a rapid decrease in the number of biofilm bacteria and associated phages, infectious phages were still detected on surfaces after completion of the 150min cleaning procedure. The resulting sequence of phage removal was: GA>Q >> MS2. ConclusionsThe effect of chlorine on biofilm bacteria and biofilm-associated phages was limited to the upper layers of the biofilm and was not enhanced by an increase in hydrodynamic shear stress. A smaller decrease was observed for MS2 than for GA or Q after completion of the cleaning procedure. Significance and Impact of the StudyThe differences observed between the three phages suggest that the location of the viral particles in the biofilm, which is related to their surface properties, affects the efficiency of chlorine disinfection.