Subterranean estuaries (STEs) form in the subsurface where fresh groundwater and seawater meet and mix. Subterranean estuaries support a variety of biogeochemical processes including those transforming nitrogen (N). Groundwater is often enriched with dissolved inorganic nitrogen (DIN), and transformations in the STE determine the fate of that DIN, which may be discharged to coastal waters. Nitrification oxidizes ammonium (NH4+) to nitrate, making DIN available for N removal via denitrification. We measured nitrification at an STE, in Virginia, USA using in situ and ex situ methods including conservative mixing models informed by in situ geochemical profiles, an in situ experiment with 15NH4+ tracer injection, and ex situ sediment slurry incubations with 15NH4+ tracer addition. All methods indicated nitrification in the STE, but the ex situ sediment slurries revealed higher rates than both the in situ tracr experiment and mixing model estimations. Nitrification rates ranged 55.0–183.16 μmol N m−2 d−1 based on mixing models, 94.2–225 μmol N m−2 d−1 in the in situ tracer experiment, and 36.6–109 μmol N m−2 d−1 slurry incubations. The in situ tracer experiment revealed higher rates and spatial variation not captured by the other methods. The geochemical complexity of the STE makes it difficult to replicate in situ conditions with incubations and calculations based on chemical profiles integrate over longer timescales, therefore, in situ approaches may best quantify transformation rates. Our data suggest that STE nitrification produces NO3−, altering the DIN pool discharged to overlying water via submarine groundwater discharge.