Eutrophication of the Chesapeake Bay has contributed to a decline in ecosystem health within the watershed. In this study, MESA (a metabolite of metolachlor) and sucralose were proposed as stable tracers that could be used to discriminate nutrient contributions from agricultural versus urban sources. Two estuaries of the Chesapeake Bay were chosen as model systems: the Choptank River (agricultural) and the Anacostia River (urban). Surface water samples were collected and analyzed for herbicides, metabolites, artificial sweeteners and nutrients. Results supported the hypothesis that sucralose is present in waterways influenced by wastewater plants, and MESA was correlated with changes in nitrate concentration in the Choptank indicating agricultural sources. This work provides proof of concept that tracers can be used in the Chesapeake Bay region to distinguish the influence of urban and agricultural nutrient loads and provides a path to better assess restoration efforts and improved allocation of total maximum daily loads.