The generation and dissemination of antibiotic resistance bacteria (ARB) and antibiotic resistance genes (ARGs) in the environment has become a critical risk to human health. This study is based on a pilot-scale simulated water distribution system to understand the effects of chlorine disinfection treatment (without free chlorine) on ARB and ARGs in biofilms. The hydraulic parameters and pipe materials of the system were simulated based on a drinking water system. The results of the colony counts showed that bacterial multi-antibiotic resistance could be enhanced 13-fold in the biofilms of the pipeline. The use of high-throughput qPCR (HT-qPCR) indicated that the total relative abundance of ARGs in biofilm samples increased significantly (p, 0.05), while the diversity of bacteria was shown to be reduced via taxonomic analysis of the V3–V4 region of 16S rRNA. The prominent types of ARGs were conferred resistance by aminoglycoside and β-lactam after the chlorine disinfection treatment, and antibiotic deactivation was the main mechanism. Phyla Proteobacteria had the highest abundance in both treatment and control groups but decreased from 70.81% (initial biofilm sample) to 26.09% (the sixth-month biofilm sample) in the treatment groups. The results show that the chlorine disinfection plays a role in the risk of development of bacterial antibiotic resistance in pipe networks owing to bacteria in biofilms. This study was the first to investigate the contribution of chlorination without free chlorine to the bacterial community shift and resistome alteration in biofilms at a pilot-test level.