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This dataset contains nitrogen (N) fixation and isotope data from experimental samples collected at Toolik Lake, Alaska during the 2022 and 2023 growing seasons. Sampling was conducted across multiple block treatments to capture spatial variability and included four substrate types: lichen, moss, litter, and soil. Within each plot, substrates were collected systematically along transects to ensure representative sampling, with lichen samples collected opportunistically due to lower abundance. Samples were incubated in the field under ambient conditions using 15N₂ to measure biological nitrogen fixation (BNF). In 2023, a short-term wetting experiment was conducted to assess the influence of moisture on BNF rates, with subsamples exposed to controlled additions of water. Across both years, data include isotope ratios, incubation conditions, moisture, fresh and dry biomass, and treatment assignments. The dataset provides information on BNF across substrate types, moisture regimes, and fertilization treatments in Arctic tundra. These data support investigation of N cycling processes, the influence of moisture and fertilization on fixation rates, and variability across vegetation types. The dataset is complete for the two field seasons (2022 and 2023) and includes sample- and block-level metadata necessary for reuse in ecological and biogeochemical research.