Hydrodynamics impacts interactions between microbes and their micro-habitats in aqueous systems, thus the study of hydrodynamics is key to understanding the formation and dynamics of biofilms. Yet mechanisms of how microbial responses to hydrodynamics regulate biofilm formation in drinking water distribution systems (DWDS) are underappreciated. Here, we investigated the linkage between early-stage biofilm formation and flow velocity fluctuations in a model DWDS. Results showed that an intermediate velocity (1.0 m/s) enhanced biofilm formation, with the highest biofilm/total cells ratio of 96.91% ± 2.26%. Moreover, the intermediate velocity promoted extracellular polymeric substances (EPS) release, accompanied with lowered zeta potential and elevated hydrophobicity of suspended cells, which could be responsible for surface aggregation. Shifts in biofilm community were observed along with hydrodynamics fluctuations. Intermediate velocity (1.0 m/s) stimulated the dominance of Proteobacteria (78.16%) along with the genus predominance of Pseudomonas, known to secrete large amounts of EPS favoring biofilm formation. Overall, this study provides new understanding of biofilm formation responding to hydrodynamic fluctuations in DWDS.