Systematic studies on the assembly process and drivers of biofilm microbiota communities are still limited. In this study, we used the artificial concrete channel of the world's largest interbasin water diversion project, the middle route of the South-to-North Water Diversion Project in China, as a model system to investigate the assembly mechanisms of biofilm microbiota communities. Our study revealed that water temperature (p <
0.001) and hydrodynamic disturbance (p <
0.05) significantly influenced biofilm biomass. The bacterial communities exhibited substantial spatial heterogeneity, whereas the eukaryotic communities presented pronounced spatial and seasonal variations (PERMANOVA, p <
0.05). Neutral model and null model analyses indicated that dispersal limitation and homogeneous selection (54.8 %-69.7 % in bacteria and 55.9 %-76.1 % in eukaryotes) predominantly governed community assembly. Deterministic effects such as hydrodynamic conditions and temperature strongly influence eukaryotes (homogeneous selection accounts for 63.9 % of eukaryotes in spring). The metacommunity network could be divided into five primary modules with key nodes, including many species from Proteobacteria, Chlorophyta, Bacillariophyta, and Cyanobacteria. Bacteria, such as Proteobacteria, Chlorophyta, Cyanobacteria, and Bacteroidota, act as connectors and a vital role in maintaining the coexistence of modules. Finally, we confirmed that physicochemical (hydrodynamic conditions, temperature, dissolved oxygen conductivity permanganate index), spatial, and biological factors have significant effects on both bacterial and eukaryotic communities as well as metacommunity networks. Our findings provide new insights into the different assembly processes and drivers of bacterial and eukaryotic communities in biofilms, which is highly important for water quality monitoring and sustainable water diversion.