Summary1. International river catchments pose challenges for effective water resource management. Catchment‐wide strategies are often complicated by differences in national bioassessment and quality classification. Intercalibration efforts aim to harmonise these differences, but rely on the consistent delineation of near‐natural reference sites that are almost unavailable in today’s landscape, especially for large rivers.2. We introduce the concept of alternative benchmarking that is based on the notion of aquatic communities at similar (low) levels of impairment associated with least‐disturbed conditions (LDC) as defined by abiotic criteria. Using data acquired during the second Joint Danube Survey, we defined LDC sites based on a multivariate gradient of anthropogenic pressures, mostly related to morphological deterioration, that spans the entire navigable Danube.3. The river was subdivided into four stretches, each featuring homogeneous biological assemblages. Indirect gradient analysis revealed relationships between the pressure gradient and selected features of the macroinvertebrate and macrophyte community but not for diatoms or phytoplankton.4. We identified biological metrics suitable for the quality classification of individual stretches or the entire river. Impoundment is the major hydromorphological alteration on the Danube but various metrics still responded significantly to differences in the morphological condition of sites not affected by impoundment.5. A comparison of macroinvertebrate sampling techniques (airlift versus kick‐and‐sweep) revealed differences in how the acquired data reflect the effects of anthropogenic pressure. Biological metrics based only on kick‐and‐sweep sample data were insensitive to habitat deterioration in the heavily modified Upper Danube.6. This study exemplifies the empirical approach of alternative benchmarking in intercalibration and offers practical solutions to some of the challenges of large river bioassessment.