ABSTRACTOf all ecosystems, freshwaters support the most dynamic and highly concentrated biodiversity on Earth. These attributes of freshwater biodiversity along with increasing demand for water mean that these systems serve as significant models to understand drivers of global biodiversity change. Freshwater biodiversity changes are often attributed to hydrological alteration by water‐resource development and climate change owing to the role of the hydrological regime of rivers, wetlands and floodplains affecting patterns of biodiversity. However, a major gap remains in conceptualising how the hydrological regime determines patterns in biodiversity's multiple spatial components and facets (taxonomic, functional and phylogenetic). We synthesised primary evidence of freshwater biodiversity responses to natural hydrological regimes to determine how distinct ecohydrological mechanisms affect freshwater biodiversity at local, landscape and regional spatial scales. Hydrological connectivity influences local and landscape biodiversity, yet responses vary depending on spatial scale. Biodiversity at local scales is generally positively associated with increasing connectivity whereas landscape‐scale biodiversity is greater with increasing fragmentation among locations. The effects of hydrological disturbance on freshwater biodiversity are variable at separate spatial scales and depend on disturbance frequency and history and organism characteristics. The role of hydrology in determining habitat for freshwater biodiversity also depends on spatial scaling. At local scales, persistence, stability and size of habitat each contribute to patterns of freshwater biodiversity yet the responses are variable across the organism groups that constitute overall freshwater biodiversity. We present a conceptual model to unite the effects of different ecohydrological mechanisms on freshwater biodiversity across spatial scales, and develop four principles for applying a multi‐scaled understanding of freshwater biodiversity responses to hydrological regimes. The protection and restoration of freshwater biodiversity is both a fundamental justification and a central goal of environmental water allocation worldwide. Clearer integration of concepts of spatial scaling in the context of understanding impacts of hydrological regimes on biodiversity will increase uptake of evidence into environmental flow implementation, identify suitable biodiversity targets responsive to hydrological change or restoration, and identify and manage risks of environmental flows contributing to biodiversity decline.