Bacteria play a very important role in the material circulation and energy flow of lake ecosystem. In this study, high-throughput sequencing technology and multivariate statistical analysis were used to investigate the bacterial community in Nanjing Xuanwu Lake and its inflow/outflow channels, and the main environmental factors affecting the bacterial community composition were analyzed. The results showed that there were different α diversity of bacteria community in Xuanwu Lake and the inflow/outflow channels. At the phyla level, the bacterial communities in Xuanwu Lake and its inflow/outflow channels were mainly composed of Cyanobacteria, Actinobactria, Firmicutes, Proteobacteria, Patescibacteria, Verrucomicrobia, and Planctomycetes. Cyanobacteria, Firmicutes and Proteobacteria were the most abundant phyla in the lake, outflow channels and inflow channels, respectively. At the generic level, it consisted mainly of Cyanobium PCC-6307, hgcI clade, Terrimicrobium, CL500-29 marine group, Defluviitalea, and Ruminiclostridium. Cyanobium PCC-6307, Defluviitalea and Ruminiclostridium were the most abundant genus in the lake, outflow channels and inflow channels, respectively. Typical correspondence analysis showed that TN, TDN, NO3−–N and pH were the most significant environmental factors affecting the bacterial community in Xuanwu Lake and its channels. The research results could provide theoretical evidences for the aquatic ecosystem security and environmental management of Nanjing Xuanwu Lake.