Recent developments in water resource monitoring have increased the demand for the reliable identification of faecal pollution sources, also defined as microbial (faecal) source tracking (MST). Standardized faecal indicator bacteria (SFIB) enumeration does not directly support MST, as SFIB occur in animal and human sources. The aim of this study was to rigorously evaluate the applicability of host-associated faecal genetic MST markers detected by quantitative PCR (qPCR) at representative Austrian water resources (ground-, surface-, raw and treated wastewater, n = 196 samples) with high importance for the water management sector. Groundwater covered a gradient of non- (i.e., deep wells) to surface influenced resources (i.e., karst and shallow wells). In addition, single faecal excreta from humans as well as representative livestock and wildlife species were collected to evaluate the faecal source-specificity and -sensitivity of the MST assays. Genetic MST marker resistance against UV irradiation was evaluated in on-site ground and wastewater treatment installations. Bacteroides-based human- (HF183II, BacHum), ruminant- (BacR), and pig-associated (Pig2Bac) MST marker qPCR quantification was performed in concert with cultivation of E. coli, intestinal enterococci, and Clostridium perfringens (SFIB diagnostics). The selected MST makers revealed high faecal source identification capacity for the Austrian water compartments and quantitatively reflected the selected faecal pollution gradient. The study also demonstrated that SFIB data can efficiently be combined with MST data to solve previously unanswered questions in water safety monitoring and management (e.g., support pollution source-targeted catchment protection, hazard assessment, and health risk management). Further research and development needs are discussed to exploit the full power of MST technology. In conclusion, this study illustrates the capacity of molecular faecal pollution diagnostics to revolutionize water quality testing in the decades to come.