AbstractAccurate characterisation of ecological communities with respect to their biodiversity and food-web structure is essential for conservation. However, combined empirical study of biodiversity and multi-trophic food webs at a large spatial and temporal resolution has been prohibited by the lack of appropriate access to such data from natural systems. Here, we assessed biodiversity and food-web characteristics across a 700 km² riverine network over seasons using environmental DNA. We found contrasting biodiversity patterns between major taxonomic groups. Local richness showed statistically significant, season-dependent increases and decreases towards downstream location within the catchment for fish and bacteria, respectively. Meanwhile, invertebrate richness remained spatially unchanged but varied across seasons. The structure of local food webs, such as link density and nestedness, also varied across space and time. However, these patterns did not necessarily mirror those observed for biodiversity and functional feeding characteristics. Our results suggest that biodiversity patterns and food-web dynamics are not directly scalable to each other even at the same spatial and temporal scales. In order to conserve species diversity as well as the functional trophic integrity of communities, patterns of biodiversity and food-web characteristics must thus be jointly studied.