Studies in our laboratory leaded to the suggestion of human adenoviruses (HAdV) and human polyomaviruses, specifically JC polyomavirus (JCPyV), porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as new indicators of fecal/urine contamination and as microbial source tracking (MST) tools. In this study, quantitative PCR (qPCR) assays specifically targeting HAdV, JCPyV, PAdV and BPyV have been evaluated for their specificity and concentration in a diversity of environmental samples including raw sewage for small and medium size hospitals and groundwater. A very easy low cost assay based on direct flocculation procedures has been used for the concentration of viruses and the study of samples potentially presenting mixed sources of contamination as river and ground water samples. The results of this and previous studies showed that adenoviral and polyomaviral infections are specific and excreted all over the year in all geographical areas, supporting strongly the use of these specific viruses as indicators of fecal contamination and as MST tools. Ground water samples from areas presenting high levels of nitrates were successfully analyzed using the described tools in order to identify potential sources of contamination. The cost-effective and robust methods described for the quantification of viruses in water provide sensitive analytical tools with which to study viruses in water and develop new standards for improving the control of the microbiological quality of food and water, to trace the origin of fecal contamination, and to assess the efficiency of virus removal in water treatment plants.