The lack of data from non-perennial rivers, which regularly cease to flow and dry up, poses a significant challenge in understanding river biodiversity. These dynamic ecosystems, accounting for over half of the global river network, remain understudied. To address this gap, we conducted a coordinated experiment and a metabarcoding approach on environmental DNA targeting multiple taxa (i.e. Archaea, Bacteria, Fungi, Algae, Protozoa, Nematoda, Arthropoda and Streptophyta). Dry sediments were collected from 84 non-perennial rivers across 19 countries on five continents to investigate biodiversity patterns and drivers. Our findings reveal that both direct (e.g., nutrient and carbon availability) and indirect (e.g., latitude and climate) drivers significantly influence local biodiversity for most taxa. In particular, we observed that low resource availability and extended dry phases promote the prevalence of oligotrophic microbial taxa, similar to soil microbial communities during drought events. Interestingly, our inferred partial correlation networks indicate that covariation among taxa, particularly Bacteria, Fungi, Algae, and Protozoa, plays a more substantial role in explaining variations in community composition than abiotic gradients. This unexpected result suggests that biotic interactions may drive community assembly in non-perennial rivers, potentially shaping biodiversity responses in the face of global changes like warming and altered hydrological patterns.