Poor sanitation in rural infrastructure is often associated with high levels of fecal contamination in adjacent surface waters, which presents a community health risk. Although microbial source tracking techniques have been widely applied to identify primary remediation needs in urban and/or recreational waters, use of human-specific markers has been more limited in rural watersheds. This study quantified the human source tracking marker Bacteroides-HF183, along with more general fecal indicators (i.e. culturable Escherichia coli and a molecular Enterococcus marker), in two Appalachian streams above and below known discharges of untreated household waste. Although E. coli and Enterococcus were consistently recovered in samples collected from both streams, Bacteroides-HF183 was only detected sporadically in one stream. Multiple linear regression analysis demonstrated a positive correlation between the concentration of E. coli and the proximity and number of known waste discharge points upstream; this correlation was not significant with respect to Bacteroides-HF183, likely due to the low number of quantifiable samples. These findings suggest that, while the application of more advanced source targeting strategies can be useful in confirming the influence of substandard sanitation on surface waters to justify infrastructure improvements, they may be of limited use without concurrent traditional monitoring targets and on-the-ground sanitation surveys.