Biofilms were cultivated for a 68-day period under different hydrodynamic conditions, and the effect of hydrodynamics on the succession of autotrophic and heterotrophic organisms of biofilms was investigated. Five obvious stages were observed during biofilm formation. At Stage I, the attachment of algae was delayed, especially under turbulent conditions. After Stage II, algal density and heterotrophic biomass of biofilms increased, which were obvious under turbulent flow. Therefore, the algal density and heterotrophic biomass of biofilms were largest under turbulent condition, followed by laminar condition, and then transitional condition. Diatoms were dominant in all flumes and were most abundant under turbulent conditions. The proportion of cyanobacteria was highest under laminar conditions. The ratio of aerobic to anaerobic bacteria decreased and their co-existence could facilitate the nitrification and denitrification in the biofilm. The ratio of monounsaturated fatty acids to saturated fatty acids was highest under turbulent conditions on the 15th day. While the ratio was highest under laminar condition on the 48th day, the high ratio indicates the high ability of biofilm to obtain nutrients, which affect the growth of algae. The regulation of hydrodynamics is a useful technology which can affect the growth of the microorganisms of biofilm, and further improve water quality.