<p>Biologically provided nitrogen (from diet or symbionts) is overwhelmingly vital to animal life on Earth. Biological nitrogen provisioning (BNP), mainly via nitrogen fixation and recycling, is a well-established function of the gut microbiomes of various terrestrial insects but remains less well-studied in freshwater insects. One possible route for BNP in freshwater insects is microbial nitrate reduction to ammonium (NRA), which produces the same intermediate metabolite, ammonium, as microbial nitrogen fixation in terrestrial insects. In this study, we characterized gut microbiome composition using 16S amplicon sequencing, followed by compound-specific stable isotope analyses (CSIA), to examine gut microbial NRA as a possible function in nymphs of the herbivorous, filter-feeding mayfly (<i>Hexagenia bilineata</i>) and the predatory dragonfly (<i>Tetragoneuria spinigera)</i>. We first uncovered significant differences in microbiome community composition and elevated NRA potential in mayflies compared with dragonflies. We subsequently determined significantly greater ¹⁵N-signatures across essential amino acids (δ¹⁵N_EAA) and non-essential amino acids (δ¹⁵N_NEAAs) in the herbivorous mayfly than the predatory dragonfly, after incubation in K¹⁵NO₃ for 5 days. This is interpreted as insect host uptake and incorporation of microbe-derived ¹⁵NH₄ into amino acids following NRA. Taken together, the results presented here suggest that gut microbial NRA may be a potential mechanism for supplying biological nitrogen to freshwater insects, potentially helping alleviate nitrogen limitations. This work advances our understanding of freshwater insect host-gut microbe associations (nature and function) and the potential impacts of these associations on host and ecosystem processes.</p>