On-site wastewater systems are commonly used to treat wastewater in rural areas, and many studies have shown these systems to be efficient at reducing phosphorus concentrations. However, as rural areas urbanize and the density of on-site systems increases, effluent discharges to the subsurface may contribute to water quality degradation. Many residences (~50%) use on-site systems in North Carolina where excess nutrient loadings have caused water use impairment. The goal of this study was to determine whether on-site system inputs had a measurable influence on stream phosphorus concentrations and loads. Seven watersheds including 3 served by on-site systems and 4 control watersheds served by a municipal sewer system in coastal North Carolina were sampled each month for 13 months during baseflow conditions and during two storm events for total phosphorus and total dissolved phosphorus analyses and physical and chemical characterization. Watersheds served by on-site systems had significantly (p < 0.05) larger concentrations of total phosphorus and total dissolved phosphorus relative to the control watersheds (municipal sewer) that did not receive wastewater discharges. Mass loads of total phosphorus and total dissolved phosphorus were also significantly (p < 0.05) greater from watersheds served by on-site systems relative to municipal sewer. These data suggest that on-site systems influenced the water quality of streams with regard to in-stream phosphorus concentrations and led to higher phosphorus loading to downstream aquatic ecosystems relative to control watersheds. Watershed nutrient management plans should address phosphorus contributions from both on-site systems and municipal sewer systems in coastal plain regions.