Nitrogen (N) fixation is a fundamental mechanism by which N enters streams. Yet, because of modern N saturation, it is difficult to study the importance of N-fixation to stream nutrient budgets. Here, we utilized relatively simple and pristine McMurdo Dry Valley streams to investigate the role of N-fixing Nostoc abundance, streamwater dissolved inorganic N (DIN) concentration, and distance from the source glacier in regulating the elemental and isotopic composition of three microbial mat types (black, orange, and green) at the landscape scale. We found Nostoc-based black mats were the most enriched in δ15N, and δ15N signatures of mats increased where Nostoc was abundant, but did not surpass the atmospheric standard (δ15N ≈ 0‰). Furthermore, green and orange mat δ15N signatures became more depleted with increasing DIN, indicating that mats utilize glacial meltwater-sourced N when available. The distance from the source glacier explained limited variability in mat δ15N across sites, indicating the influence of individual stream characteristics on N spiraling. To further explore longitudinal N spiraling processes generating observed δ15Ν patterns, we developed a simple steady-state mathematical model. Analysis of plausible scenarios with this model confirmed that streams both have the capacity to remove allochthonous DIN over the plausible range of inputs, and that internal N sources are required to account for δ15N signatures and observed DIN concentrations at stream outlets. Collectively, these data and modeling results demonstrate that N-fixation exerts substantial influence within and across these streams, and is presumably dependent upon interconnected organic matter reserves, mineralization rates, and geomorphology.