AIMS: This 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 Qβ. METHODS AND RESULTS: Chlorination (4 mg l(-1) ) was applied first with a hydrodynamic shear stress of 1 Pa and second with an increase in hydrodynamic shear stress to 10 Pa. Despite a rapid decrease in the number of biofilm bacteria and associated phages, infectious phages were still detected on surfaces after completion of the 150 min cleaning procedure. The resulting sequence of phage removal was: GA > Qβ ≫ MS2. CONCLUSIONS:The 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 Qβ after completion of the cleaning procedure. SIGNIFICANCE AND IMPACT OF THE STUDY:The 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.