Aquifers provide integral freshwater resources and host ecosystems of largely uncharacterized, truncated endemic microorganisms. In recent history, many aquifers have become increasingly contaminated from various anthropogenic sources. To better understand the impacts of nitrogen contamination on native groundwater ecosystems, 16S rRNA sequencing of the groundwater microbial communities was carried out. Samples were taken from an aquifer known to be contaminated with nitrogen from multiple sources, including fertilizers and wastewater treatment plant effluents. In total, two primary contaminants were identified: NH4+ (<0.1–3.7–26 mg L−1 NH4+ minmedian-max), and NO3− (<0.01–18–150 mg L−1 NO3− min-median-max). These contaminants were found to be associated with a decrease/increase in microbial species richness within affected groundwater for NH4+/NO3−, respectively. Important phyla were identified, including Proteobacteria, which had the highest abundance within samples unaffected by NH4+ (36–81% NH4+ unaffected, 4–33% NH4+ affected), and Planctomycetes (0.05–10% NH4+ unaffected, 43–72% NH4+ affected), which had the highest abundance within the NH4+ affected samples, likely due to its ability to perform anaerobic ammonia oxidation (ANAMMOX). Planctomycetes were identified as a potential indicator for the presence of NH4+ contamination. The analysis and characterization of sequencing data alongside physicochemical data showed potential to increase the depth of our understanding of contaminant behavior and fate within a contaminated aquifer using this type of data and analysis.