Understanding natural and anthropogenic factors driving patterns of biodiversity is a fundamental goal in ecology. Despite the importance of the hydrological regime for aquatic ecosystems being well understood, we still lack a clear and unified conceptual understanding of how hydrology influences aquatic biodiversity from regional to local spatial scales. Much of our current understanding is based on the taxonomic and functional richness of communities at individual locations and how these facets of biodiversity are shaped by natural hydrological disturbances (e.g. floods, droughts) or altered by anthropogenic changes to hydrological regimes due to human water use and hydrological regulation. We review and synthesise published literature on hydrology-biodiversity relationships to (i) determine how scale-dependent components of freshwater biodiversity respond to hydrological gradients and hydrological regimes and (ii) identify the specific underlying ecohydrological mechanisms responsible for patterns of biodiversity across spatial scales. Different ecohydrological mechanisms vary in their effect on freshwater biodiversity across spatial scales; hydrological disturbance regime is an important driver of biodiversity across local-regional scales, and both hydrological connectivity and the effect of hydrology on habitat strongly influence landscape and local scale biodiversity. We highlight that there remains a lack of research to understand how hydrological regimes influence both functional and phylogenetic aspects of freshwater communities across spatial scales and identify six research priorities necessary to improve our understanding of multi-scaled biodiversity responses to hydrological regimes. Addressing these gaps in understanding is critical, as a central goal of freshwater conservation policy is to manage hydrological regimes to protect or enhance freshwater biodiversity.