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Fecal water pollution is a global problem, as it is associated with risks to human health. For more than 100 years now, so-called fecal indicator bacteria (FIB), usually E. coli and intestinal enterococci, have been used to assess microbiological water quality. However, as these bacteria are ubiquitous in the digestive tract of humans and animals, the identification of the contamination source, which plays an important role in risk assessment, is impossible. Since the early 2000s, aided by rapid advances in molecular biology methods, techniques have been developed that allow targeted source detection via host-associated markers. These MST (microbial source tracking) methods are increasingly used for the analysis and modeling of microbial hazards and risks to support decision-making in water management. In addition, the feasibility of organic micropollutants for risk assessment (chemical source tracking) has been discussed. However, there are currently no comprehensive studies that have systematically compared the performance of these parameters along the full fecal pollution gradient (groundwater to untreated wastewater). In this study, a unique sample set was compiled and examined, covering the entire fecal contamination gradient for the first time. It included untreated and conventionally treated municipal wastewaters, various surface waters, and a broad set of porous groundwater resources covering a gradient from non-influenced (i.e., deep wells) to surface-influenced resources (i.e., shallow wells).The results showed that the human-associated contamination gradient could be accurately represented using cultivation-based FIB markers (E. coli, enterococci). No FIB were detected in deep wells or wells, sporadic detections were encountered in surface influenced wells and continuous detections in surface waters and raw and treated wastewater. FIBs determined by molecular techniques (qPCR) corresponded well with those of the cultivation-based methods. All genetic markers used (human-associated: BacHum, HF183/BacR287, wastewater associated: Lachno3) also accurately represented the gradient, but the concentrations detected were higher than those for the classical FIB. This is due to their significantly higher concentrations in feces and represents a clear advantage for detection in the environment. In contrast, the mitochondrial marker (mtDNA_hum) appeared in significantly lower concentrations compared to the bacterial markers. Of the 37 investigated chemical substances, it was mainly small traces of artificial sweeteners that could be detected even in some deep wells. In rivers, treated and raw wastewater nearly all determined compounds (predominantly pharmaceuticals) were detected. Regarding concentrations of these tracers the pollution gradient also became apparent. To summarize, applicable genetic MST markers follow the same gradient as standardized FIB and are equally sensitive (no false positives in no-influenced habitats). They exhibit comparable sample limits of detection in equivalent sample volumes, but with the advantage that they can directly track the municipal wastewater path (source specific detection of human fecal pollution). This makes them an ideal complement to the FIB approach. Chemical tracers can be a useful aid, but they must be chosen carefully because, unlike genetic MST markers, they do not occur ubiquitously in municipal raw sewage (primary sources of pollution). Regional differences due to the use of different therapeutic agents play a major role.