Understanding the drivers of species distribution is an important topic in conservation biology and ecology, pertaining to species traits like dispersal strategies and species–environment interactions. Here we examined the drivers of benthic species distribution at 20 sections of a second-order stream network. Environmental and spatial factors and the dispersal modes of the organisms were considered. We expected that species with aerial dispersal capabilities like insects would be less restrained by distance between sites and thus mostly affected by environmental factors. In contrast, we hypothesized that completely benthic species would mainly be affected by spatial factors due to limited dispersal. However, microscopic species like nematodes characterized by a high passive dispersal potential may be less limited by spatial factors. When using redundancy analyses and subsequent variance partitioning, the included variables explained 24% (insects), 24% (non-flying macrobenthos), and 32% (nematodes) of the variance in the respective community composition. Spatial factors mainly explained the species composition of all tested groups. In contrast with other larger species, nematodes were characterized by fine-scale patterns that might have been induced by random processes (e.g., random distribution and priority effects). Our study showed that dispersal processes are crucial in shaping benthic communities along streams albeit the relatively small sampling area (max. distance between sampling sites: 2 km). The demonstration of spatial factors as important drivers of the species distribution of passively dispersing benthic organismal groups highlights the role played by connectivity in determining species distribution patterns in river systems.