This work addresses multiple human stressors and their impacts on fish assemblages of the Drava and Mura rivers in southern Austria. The impacts of single and multiple human stressors on riverine fish assemblages in these basins were disentangled, based on an extensive dataset. Stressor configuration, i.e. various metrics of multiple stressors belonging to stressor groups hydrology, morphology, connectivity and water quality were investigated for the first time at river basin scale in Austria. As biological response variables, the Fish Index Austria (FIA) and its related single as well as the WFD biological- and total state were investigated. Stressor-response analysis shows divergent results, but a general trend of decreasing ecological integrity with increasing number of stressors and maximum stressor is observed. Fish metrics based on age structure, fish region index and biological status responded best to single stressors and/or their combinations. The knowledge gained in this work provides a basis for advanced investigations in Alpine river basins and beyond, supports WFD implementation and helps prioritizing further actions towards multi-stressor restoration- and management.