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A model biofilm, formed of multiple species from environmental drinking water, including opportunistic pathogens, was created to explore the tolerance of multi-species biofilms to chlorine levels typical of water-distribution systems. All species, when grown planktonically, were killed by concentrations of chlorine within the World Health Organization guidelines (0.2–5.0 mg l−1). Higher concentrations (1.6–40-fold) of chlorine were required to eradicate biofilm populations of these strains, ∼70% of biofilms tested were not eradicated by 5.0 mg l−1 chlorine. Pathogenic bacteria within the model multi-species biofilms had an even more substantial increase in chlorine tolerance; on average ∼700–1100 mg l−1 chlorine was required to eliminate pathogens from the biofilm, 50–300-fold higher than for biofilms comprising single species. Confocal laser scanning microscopy of biofilms showed distinct 3D structures and multiple cell morphologies and arrangements. Overall, this study showed a substantial increase in the chlorine tolerance of individual species with co-colonization in a multi-species biofilm that was far beyond that expected as a result of biofilm growth on its own.