Nitrate (NO₃⁻) concentrations and dual isotopic composition (δ¹⁵N and δ¹⁸O) were measured during various seasons and tidal conditions in Elkhorn Slough to evaluate mixing of sources of NO₃⁻ within this California estuary. We found the isotopic composition of NO₃⁻ was influenced most heavily by mixing of two primary sources with unique isotopic signatures, a marine (Monterey Bay) and terrestrial agricultural runoff source (Old Salinas River). However, our attempt to use a simple two end‐member mixing model to calculate the relative contribution of these two NO₃⁻ sources to the Slough was complicated by periods of nonconservative behavior and/or the presence of additional sources, particularly during the dry season when NO₃⁻ concentrations were low. Although multiple linear regression generally yielded good fits to the observed data, deviations from conservative mixing were still evident. After consideration of potential alternative sources, we concluded that deviations from two end‐member mixing were most likely derived from interactions with marsh sediments in regions of the Slough where high rates of NO₃⁻ uptake and nitrification result in NO₃⁻ with low δ¹⁵N and high δ¹⁸O values. A simple steady state dual isotope model is used to illustrate the impact of cycling processes in an estuarine setting which may play a primary role in controlling NO₃⁻ isotopic composition when and where cycling rates and water residence times are high. This work expands our understanding of nitrogen and oxygen isotopes as biogeochemical tools for investigating NO₃⁻ sources and cycling in estuaries, emphasizing the role that cycling processes may play in altering isotopic composition.