Fecal matter is considered as one of the worst pollutants in waterbodies due to the potential spread of waterborne diseases. This study aimed to determine the host-specific fecal contamination in two Brazilian watersheds and to predict the possible impacts on human health. Fecal sources were enumerated using host-specific genetic markers to swine (16S rRNA), human and bovine (archaeal nifH), and equine (archaeal mcrA). A single cycling condition was established for four markers aiming to decrease the analysis time. Fifteen samples from São João watershed (75%) and 25 from Guandu (62.5%) presenting Escherichia coli enumeration in compliance with Brazilian guidelines (<1,000 MPN/100 mL) showed the human marker. Furthermore, the bovine, swine, and equine markers were present in 92% (59/64), 89% (57/64), and 81% (52/64) of the water samples, respectively. The molecular markers proposed for qPCR in our study were sensitivity and specific enough to detect host-specific fecal pollution in all samples regardless of E. coli levels reaffirming the low correlation among them and supporting their use in water quality monitoring programs. To our knowledge, this is the first study using this approach for quantification of nifH, mcrA, and rrs gene-associated human and animal fecal pollution in waters intended for drinking water supply in Brazil.