Anaerobic ammonium oxidation (anammox) couples the oxidation of ammonium with the reduction of nitrite, producing N₂. The presence and activity of anammox bacteria in groundwater were investigated at multiple locations in an aquifer variably affected by a large, wastewater-derived contaminant plume. Anammox bacteria were detected at all locations tested using 16S rRNA gene sequencing and quantification of hydrazine oxidoreductase (hzo) gene transcripts. Anammox and denitrification activities were quantified by in situ ¹⁵NO₂–tracer tests along anoxic flow paths in areas of varying ammonium, nitrate, and organic carbon abundances. Rates of denitrification and anammox were determined by quantifying changes in ²⁸N₂, ²⁹N₂, ³⁰N₂, ¹⁵NO₃–, ¹⁵NO₂–, and ¹⁵NH₄+ with groundwater travel time. Anammox was present and active in all areas tested, including where ammonium and dissolved organic carbon concentrations were low, but decreased in proportion to denitrification when acetate was added to increase available electron supply. Anammox contributed 39–90% of potential N₂ production in this aquifer, with rates on the order of 10 nmol N₂–N L^–1 day^–1. Although rates of both anammox and denitrification during the tracer tests were low, they were sufficient to reduce inorganic nitrogen concentrations substantially during the overall groundwater residence times in the aquifer. These results demonstrate that anammox activity in groundwater can rival that of denitrification and may need to be considered when assessing nitrogen mass transport and permanent loss of fixed nitrogen in aquifers.