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Abstract Nitrogen isotope (δ¹⁵N) values in ancient rocks have been used to interpret the presence of nitrogen metabolisms and fixed N availability across the Archean and Paleoproterozoic eons. However, how δ¹⁵N signals produced by nitrogen metabolisms of microbial communities, the impact of the geochemical environments they live in on those signals, and the fidelity of those signals through preservation in the rock record have not been fully constrained and validated. Thus, it is imperative to study modern microbial systems to test the validity of using δ¹⁵N signals produced by microbial communities to interpret what geochemical environments and nitrogen metabolisms influenced the production of those signals. Hydrothermal systems are an ideal place to examine the biotic and abiotic factors that impact δ¹⁵N signals—physical processes generate geochemical environments with wide ranges of fixed N availability and the physicochemical environments exclude multicellular eukaryotic organisms. Previous work has demonstrated the presence of nitrogen fixation genes in microbial communities across a range of temperature (16–89°C) and pH (1.9–9.8) gradients. Here, we test the validity and fidelity of using microbial community δ¹⁵N signals as indicators of geochemical environment and nitrogen metabolisms (specifically, biological nitrogen fixation) present in eight hydrothermal systems across Yellowstone National Park. Our results suggest that δ¹⁵N values measured in the ancient rock record can provide information about the N cycling and prevailing environmental conditions during deposition, but only if viewed within appropriate context.