Non-perennial streams have a distinct hydrology governed by alternating drying and rewetting cycles that strongly shape biogeochemical cycling. However, the effects of shifting hydrological regimes on nitrogen cycling processes (e.g., N2 fixation and denitrification) remain understudied. Our study addresses this knowledge gap by examining the spatial and temporal variability of nitrogen dynamics within the South Fork of King’s Creek in the Konza Prairie Biological Station (Kansas, USA). We conducted watershed-scale synoptic sampling of surface water at 50 headwater sites (1-3rd order), 42 of which had water present for sampling. To capture seasonal variability, seven of the sites were subsequently sampled across spring, summer, and fall in 2022 and 2023. We used membrane inlet mass spectrometry (MIMS) to quantify N2 excess, which can indicate net N2-fixing or denitrifying conditions. Additional water samples were collected to measure physicochemical variables and characterize microbial community composition (16S rRNA). N2 excess signatures varied spatially, with most sites indicating net N2 fixation conditions (30 sites; -0.04 ± 0.02) and fewer showing net denitrification conditions (12 sites; 0.15 ± 0.21). In contrast, all seven seasonally sampled sites exhibited N2 excess signatures indicative of net N2 fixation (-0.29 ± 0.10). No physicochemical variable was significantly correlated with N2 excess, and differential abundance analysis determined similar microbial community composition between net N2 fixation and denitrification conditions sites. Antecedent drying conditions, along with other hydrological metrics such as network connectivity and topographic wetness index, will be examined in future analyses to assess their potential effects on nitrogen dynamics. Ultimately, our study provides greater insight into the spatial and temporal variability of nitrogen cycling processes in a non-perennial stream.