<p>Despite efforts to minimize human impact on rivers, native teleost fish and lamprey species communities in Germany remain threatened. The aim of the study was to quantify current species diversity in the study area, identify the level of deviation from realistic reference conditions, and characterize relevant drivers of diversity loss. Site-specific survey data were consolidated with reference fish community data, and selected biodiversity metrics were calculated and compared for 390 river sections. A novel species deviation index was implemented that reflects the level at which a community deviates from a presumptive natural state. The obtained biodiversity data were cross-linked with environmental factors using generalized linear mixed modeling. A significant decline in biodiversity was revealed when comparing results of the recent surveys with the corresponding reference conditions. Non-native species, transverse structures, and human land use were identified as important drivers of low native species diversity. The deviation index was especially effective in quantifying changes in fish communities and revealed ongoing dynamic environmental influences, such as bioclimatic change, that do not necessarily affect biodiversity metrics but lead to significant deviation from reference conditions. Authorities and stakeholders are encouraged to establish site-specific reference data to develop more targeted species conservation measures.</p>