Septic systems are designed to treat and release partially treated wastewater into the subsurface. In doing so they release various pollutants including nutrients, pathogens, and emerging contaminants into the environment. The objectives of this study are to (1) evaluate whether the amount of septic wastewater effluent reaching streams varies seasonally and between high and low stream flow conditions, and (2) assess the utility of using the artificial sweetener acesulfame combined with the human-specific bacterial DNA marker HF183 to infer the dominant pathways in delivering septic effluent to streams. Repeated sampling of streams in 15 subwatersheds was conducted together with detailed high-resolution longitudinal sampling along one stream. The percentage of septic effluent reaching streams was found to be highly variable between subwatersheds with septic effluent inputs greater in spring compared to other seasons, and also greater during high stream flow conditions. Stream HF183 concentrations were not correlated with acesulfame concentrations with data indicating that rapid pathways may be more important for contributing septic effluent to streams in some subwatersheds compared to others. Finally, high-resolution survey data showed that spatial trends in acesulfame and HF183 concentrations along the surveyed stream were not consistent and therefore pathways contributing effluent may vary longitudinally. The study findings are needed to improve estimates of pollutant loads to streams from septic systems and to inform septic system best management practices.