Elevated levels of pathogen pose serious risks to steams and public health. In many of streams in the U.S., elevated levels of pathogen are linked to non-point source pollution (NPS). The NPS pollution of pathogen at watershed scale is mainly controlled by the complex interactions among land cover/land use, soil characteristics, elevation, stream geometry, and climate (i.e., temperature, rainfall, and solar radiation). Improving understanding of how each characteristics of a watershed potentially influences pathogen loads in streams requires watershed modeling, which can help in identifying water quality Best Management Practices (BMPs) and controlling pathogen/pathogen indicator NPS loads. Here, we have developed a model for predicting pathogen loads in stream bed sediment and the water column. Subsequently, the model was integrated with Soil and Water Assessment Tool (SWAT) model for developing a watershed scale hydrological model for estimating E. coli Total Maximum Daily Loads (TMDLs). The model predictions were verified by multiple-years monitoring of in-stream bed sediment and water column E. coli levels. We anticipate the approach of modeling and monitoring proposed here will help improving existing understanding of in-stream pathogen contamination.