A central question for ecologists is to understand processes structuring species assemblages. Environmental and dispersal-related processes interplay, leading to potential species replacement (turnover) or species loss (nestedness) patterns. However, the relative contribution of these processes is still unclear, especially in freshwater ecosystems. Here, we examined the relative influence of environmental filtering, mass effects and dispersal limitations along the watercourse on diatom taxonomic and functional beta diversity. We used a random sample selection to examine the variation of nestedness and turnover components according to watercourse position of 2194 sites throughout France. We tested the variation of taxonomic and functional beta-diversity explained by pure environmental and spatial factors and correlated beta diversity values to environmental variables indicative of anthropogenic effects. The turnover component of beta diversity was largely predominant, while decreasing from up- to downstream following the total beta diversity dynamic. Environmental conditions seemed to exert a major influence on beta-diversity patterns, in particular anthropogenic factors tended to homogenize communities whatever the position along the watercourse. The influence of spatial factors increased along the longitudinal gradient. The functional beta diversity significantly changed along the watercourse and its relation with pure environment and spatial factors appeared to be guild dependent. We concluded that environmental filtering has a major influence in structuring diatoms metacommunities regardless of the watercourse position of the sites. The increasing influence of spatial factors mid- and downstream associated with results of taxonomic and functional compositions also suggest an important role of mass-effect processes at the end of the longitudinal gradient.