Drinking water distribution networks are critical infrastructures, being the potential targets for terrorists’ attacks. This study focuses at the decontamination of a drinking water distribution system after its deliberate contamination with spores of Bacillus spp. Shock chlorination, acid and alkaline flushing processes were tested for the ability to inactivate Bacillus spores in a large-scale pilot loop. The results of the study show that shock chlorination is very efficient against Bacillus spores in water; however, additional treatment is necessary to inactivate the spores adsorbed to the surfaces. A novel approach to alternation of chlorine and the biofilm release agent (NaOH) gave the best results in inactivation of Bacillus spores on PVC pipe surfaces.