Water residence time in the middle course of rivers is often too short to allow substantial phytoplankton development, and primary production is essentially provided by benthic phototrophic biofilms. However, cells occurring in the water column might derive from biofilm microalgae, and, reciprocally, sedimenting microalgae could represent a continuous source of colonizers for benthic biofilms. A comparative study of biofilm and pelagic microphytic communities (with special focus on diatoms) was carried out over 15 mo in the Garonne River, France. Diatoms dominated both biofilm and pelagic microphytic communities. Typically benthic diatoms were found in high abundance in the water column, and their biomass in the water was correlated with their biomass in the biofilm, indicating the benthic origin of these cells. Variations in river discharge and temperature drove the temporal distribution of benthic and pelagic communities: under high flow mixing (winter) communities showed the greatest similarity, and during low flow (summer)they differed the most. Even during low flow, typical benthic species were observed in the water column, indicating that benthic−pelagic exchanges were not exclusively due to high water flow. Moreover, during low flow periods, planktonic diatoms typically settled within biofilms, presumably because of higher water residence times, and/or upstream reservoir flushing.