Biofilms are the main carrier of microbial communities throughout drinking water distribution systems (DWDSs), and strongly affect the safety of drinking water. Understanding biofilm formation potential and chlorine resistance is necessary for exploring future disinfection strategies and preventing water-borne diseases. This study investigated biofilm formation of five bacterial strains isolated from a simulated DWDS at different incubation times (24 h, 48 h, and 72 h), then evaluated chlorine resistance of 72 h incubated biofilms under chlorine concentrations of 0.3, 0.6, 1, 2, 4, and 10 mg L⁻¹. All five bacterial strains had biofilm formation potential when incubated for 72 h. The biofilm formation potential of Acinetobacter sp. was stronger than that of Bacillus cereus, Microbacterium sp. and Sphingomonas sp. were moderate, and that of Acidovorax sp. was weak. In contrast, the order of chlorine resistance was Bacillus sp. > Sphingomonas sp. > Microbacterium sp. > Acidovorax sp. > Acinetobacter sp. Thus, the chlorine resistance of a single-species biofilm has little relation with the biofilm formation potential. The biofilm biomass is not a major factor affecting chlorine resistance. Moreover, the chlorine resistance of a single-species biofilm is highly related to the physiological state of bacterial cells, such as their ability to form spores or secrete extracellular polymeric substances, which could reduce the sensitivity of the single-species biofilm to a disinfectant or otherwise protect the biofilm.