The development of benthic cyanobacteria currently raises concern worldwide because of their potential to produce toxins. As a result, understanding which measures of biotic and abiotic parameters influence the development of cyanobacterial assemblages is of great importance to guide management actions. In this study, we investigate the relative contributions of abiotic and biotic parameters that may drive the development of cyanobacterial assemblages in river biofilms. First, a 2D hydrodynamic model allowed us to retrace changes in depths and velocities according to discharge at a 4 m2 resolution. From this model, we set up three hydraulic zones in each of the 4 reaches investigated along a 50-km-long river stretch. We further used univariate, multivariate and variance partitioning analyses to assess the contribution of past and present hydraulics, present physical and chemical parameters and algae to the temporal variability of cyanobacterial assemblage composition. The cyanobacterial assemblages were generally dominated by Phormidium sp., Lyngbya sp., Planktolyngbya sp. and Oscillatoria sp., four genera known to contain potentially toxic species. The highest biovolumes of cyanobacteria were present in low velocity zones in early summer and shifted to high velocity zones in late summer, highlighting the major influence of hydraulic parameters on benthic cyanobacteria settlement and development in rivers. Considering the identified genera, biofilms present a potentially high risk of toxin production. Relations between cyanobacterial development, toxin production and environmental parameters need to be further assessed to better estimate this risk.