The Three Gorges Reservoir (TGR), formed by China's Yangtze Three Gorges Project, is the largest lake in the world, but there is too little information available about fecal contamination and waterborne pathogen impacts on this aquatic ecosystem. During two successive 1-year study periods (July 2009 to July 2011), the water quality in Wanzhou watershed of the TGR was tested with regard to the presence of fecal indicators and pathogens. According to Chinese and World Health Organization water quality standards, water quality in the mainstream was good but poor in backwater areas. Salmonella, Enterohemorrhagic Escherichia coli (EHEC), Giardia and Cryptosporidium were detected in the watershed. Prevalence and concentrations of the pathogens in the mainstream were lower than those in backwater areas. The estimated risk of infection with Salmonella, EHEC, Cryptosporidium, and Giardia per exposure event ranged from 2.9×10-7 to 1.68×10-5, 7.04×10-10 to 2.36×10-7, 5.39×10-6 to 1.25×10-4 and 0 to 1.2×10-3, respectively, for occupational divers and recreational swimmers exposed to the waters. The estimated risk of infection at exposure to the 95% upper confidence limit concentrations of Salmonella, Cryptosporidium and Giardia may be up to 2.62×10-5, 2.55×10-4 and 2.86×10-3, respectively. This study provides useful information for the residents, health care workers and managers to improve the safety of surface water and reduce the risk of fecal contamination in the TGR.