Taihu Lake is one of the largest freshwater lakes in China, serving as an important source of drinking water; >60% of source water to this lake is provided by the Tiaoxi River. This river faces serious fecal contamination issues, and therefore, a comprehensive investigation to identify the sources of fecal contamination was carried out and is presented here. The performance of existing universal (BacUni and GenBac), human (HF183-Taqman, HF183-SYBR, BacHum, and Hum2), swine (Pig-2-Bac), ruminant (BacCow), and avian (AV4143 and GFD) associated microbial source tracking (MST) markers was evaluated prior to their application in this region. The specificity and sensitivity results indicated that BacUni, HF183-TaqMan, Pig-2-Bac, and GFD assays are the most suitable in identifying human and animal fecal contamination. Therefore, these markers along with marker genes specific to selected bacterial pathogens were quantified in water and sediment samples of the Tiaoxi River, collected from 15 locations over three seasons during 2014 and 2015. Total/universal Bacteroidales markers were detected in all water and sediment samples (mean concentration 6.22 log 10 gene copies/100 ml and 6.11 log 10 gene copies/gram, respectively), however, the detection of host-associated MST markers varied. Human and avian markers were the most frequently detected in water samples (97 and 89%, respectively), whereas in sediment samples, only human-associated markers were detected more often (86%) than swine (64%) and avian (8.8%) markers. The results indicate that several locations in the Tiaoxi River are heavily polluted by fecal contamination and this correlated well with land use patterns. Among the five bacterial pathogens tested, Shigella spp. and Campylobacter jejuni were the most frequently detected pathogens in water (60% and 62%, respectively) and sediment samples (91% and 53%, respectively). Shiga toxin-producing Escherichia coli (STEC) and pathogenic Leptospira spp. were less frequently detected in water samples (55% and 33%, respectively) and sediment samples (51% and 13%, respectively), whereas E. coli O157:H7 was only detected in sediment samples (11%). Overall, the higher prevalence and concentrations of Campylobacter jejuni, Shigella spp., and STEC, along with the MST marker detection at a number of locations in the Tiaoxi River, indicates poor water quality and a significant human health risk associated with this watercourse.