Surface waters are commonly used as source water for drinking water and irrigation. Knowledge of sources of fecal pollution in source watersheds benefits the design of effective source water protection plans. This study analyzed the relationships between enteric pathogens (Escherichia coli O157:H7, Salmonella spp., and Campylobacter spp. [C. jejuni, C. lari and C. coli]), water quality (turbidity, temperature, E. coli), and human and ruminant/cow Bacteroidales and mitochondrial DNA-based fecal source tracking (FST) markers in two source watersheds. Water samples (n=329) were collected at 10 sites (5 in each watershed) over 18 months. The human Bacteroidales marker (HF183) occurred in 9-10% of the water samples at nine sampling sites; while a forested site in the urban watershed tested negative. Ruminant/cow Bacteroidales markers (BacR and CowM2) only appeared in the rural watershed (6%). The mtDNA markers (HcytB and AcytB) showed the same pattern but were less sensitive due to lower fecal concentrations. Higher prevalences (P<0.05) of Campylobacter spp. (41 vs. 16% for the rural and urban watershed, respectively) and E. coli O157:H7 (12 vs. 3%) were observed in the rural watershed, while Salmonella spp. levels were comparable (23-28%). Densities of E. coli ≥ 100 colony-forming units (CFU) 100 mL-1 increased the odds (P<0.05) of detecting the enteric bacterial pathogens. The water turbidity levels (NTU ≥ 1.0) similarly predicted (P<0.05) pathogen presence. Storm events increased (P<0.01) pathogen and fecal marker concentrations in the waterways. The employment of multiple FST methods suggested failing onsite wastewater systems contribute to human fecal pollution in both watersheds.