About 60 % of Europe's rivers fail to meet ecological quality standards derived from biological criteria. The causes are manifold, but recent reports suggest a dominant role of hydro-morphological and water quality-related stressors. Yet, in particular micropollutants and hydrological stressors often tend to be underrepresented in multiple-stressor studies. Using monitoring data from four Federal States in Germany, this study investigated the effects of 19 stressor variables from six stressor groups (nutrients, salt ions, dissolved oxygen/water temperature, mixture toxicity of 51 micropollutants, hydrological alteration and morphological habitat quality) on three biological assemblages (fishes, macroinvertebrates, benthic diatoms). Biological effects were analyzed for 35 community metrics and quantified using Random Forest (RF) analyses to put the stressor groups into a hierarchical context. To compare metric responses, metrics were grouped into categories reflecting important characteristics of biological communities, such as sensitivity, functional traits, diversity and community composition as well as composite indices that integrate several metrics into one single index (e.g., ecological quality class).Water quality-related stressors – but not micropollutants – turned out to dominate the responses of all assemblages. In contrast, the effects of hydro-morphological stressors were less pronounced and stronger for hydrological stressors than for morphological stressors. Explained variances of RF models ranged 23–64 % for macroinvertebrates, 16–40 % for benthic diatoms and 18–48 % for fishes. Despite a high variability of responses across assemblages and stressor groups, sensitivity metrics tended to reveal stronger responses to individual stressors and a higher explained variance in RF models than composite indices. The results of this study suggest that (physico-chemical) water quality deterioration continues to impact biological assemblages in many German rivers, despite the extensive progress in wastewater treatment during the past decades. To detect water quality deterioration, monitoring schemes need to target relevant physico-chemical stressors and micropollutants. Furthermore, monitoring needs to integrate measures of hydrological alteration (e.g., flow magnitude and dynamics). At present, hydro-morphological surveys rarely address the degree of hydrological alteration. In order to achieve a good ecological status, river restoration and management needs to address both water quality-related and hydro-morphological stressors. Restricting analyses to just one single organism group (e.g., macroinvertebrates) or only selected metrics (e.g., ecological quality class) may hamper stressor identification and its hierarchical classification and, thus may mislead river management.