This study set out to evaluate the used next generation sequencing (NGS) approach in a representative and complex backwater catchment with importance as a drinking water resource. In a multi-compartment approach, water, faeces, soil, and sediment samples were investigated by 454 pyrosequencing of bacterial 16S rRNA gene community (V1-V2 region) to assess the capabilities of NGS methods (i) for the development and evaluation of environmental molecular diagnostics, (ii) for direct screening of the bulk bacterial communities, and (iii) for faecal pollution analysis in water.