A novel concept for fecal pollution analysis was applied at alluvial water resources to substantially extend the information provided by fecal indicator bacteria (FIB). FIB data were linked to river connectivity and genetic microbial source tracking (MST). The concept was demonstrated at the Danube River and its associated backwater area downstream of the city of Vienna, using a comprehensive 3-year data set (10 selected sites, n = 317 samples). Enumeration of Escherichia coli (ISO 16649-2), intestinal enterococci (ISO 7899-2) and Clostridium perfringens (ISO 14189) revealed a patchy distribution for the investigation area. Based on these parameters alone a clear interpretation of the observed fecal contamination patterns was not possible. Comparison of FIB concentrations to river connectivity allowed defining sites with dominating versus rare fecal pollution influence from the River Danube. A strong connectivity gradient at the selected backwater sites became obvious by 2D hydrodynamic surface water modeling, ranging from 278 days (25%) down to 5 days (<1%) of hydraulic connectivity to the River Danube within the 3-year study period. Human sewage pollution could be identified as the dominating fecal source at the highly connected sites by adding information from MST analysis. In contrast, animal fecal pollution proofed to be dominating in areas with low river connectivity. The selection of genetic MST markers was focusing on potentially important pollution sources in the backwater area, using human (BacHum, HF183II), ruminant (BacR) and pig (Pig2Bac) -associated quantitative PCR assays. The presented approach is assumed to be useful to characterize alluvial water resources for water safety management throughout the globe, by allocating fecal pollution to autochthonous, allochthonous, human or animal contamination components. The established river connectivity metric is not limited to bacterial fecal pollution, but can be applied to any type of chemical and microbiological contamination.