Effective disinfection measures are in urgent need due to the serious biosafety issues in domestic hot water supply system. In this study, biofilms annular reactor was applied to simulate the domestic hot water pipeline system. The effects of chlorine and chlorine dioxide on microbial inactivation in biofilm were investigated. The changes of microbial community and the surface structure of biofilm were further explored using Illumina MiSeq sequencing and scanning electron microscopy (SEM). The results show that by increasing disinfectant concentration, the microbial inactivation rate could be notably improved while the inactivation effect remained stable. 1.5 mg/L chlorine or chlorine dioxide did not achieve a good microbial inactivation effect. However, the complete inactivation of Escherichia coli, total number of bacteria and heterotrophic bacteria (HPC) were observed in the presence of 3-5 mg/L chlorine and chlorine dioxide. 3-5 mg/L chlorine and chlorine dioxide can effectively control the biofilm of hot water pipeline. The results suggest that adequate concentration of disinfectant and disinfection time are important for keeping high inactivation effect. The pipeline biofilm was high in bio-diversity. Different from those in municipal water supply system and the secondary water supply system, the predominant phyla of pipeline biofilm were Deinococcus-Thermus and Proteobacteria, as well as some detected pathogenic bacteria. The shock disinfection of chlorine or chlorine dioxide showed a different inactivation effect on each phylum. Specifically, compared with chlorine, chlorine dioxide had a better inactivation effect on pathogenic bacteria, Proteobacteria, and Chlamydiae. The results of SEM indicate that the surface structure of biofilm was effectively destroyed by chlorine and chlorine dioxide, resulting in the larger gaps, shrinkage, and shedding of biofilm. Moreover, the effect of chlorine dioxide on biofilm surface structure was higher than chlorine. Overall, the results are important for the biosafety in hot water system.