Arctic and subarctic soils are typically characterized by low nitrogen (N) availability, suggesting N-limitation of plants and soil microorganisms. Climate warming will stimulate the decomposition of organic matter, resulting in an increase in soil nutrient availability. However, it remains unclear how soil microorganisms in N-limited soils will respond, as the direct effect of inorganic N addition is often shown to inhibit microbial activity, while elevated N availability may have a positive effect on microorganisms indirectly, due to a stimulation of plant productivity. Here we used soils from a long-term fertilization experiment in the Subarctic (28 years at the time of sampling) to investigate the net effects of chronic N-fertilization (100 kg N ha−1 y−1, added together with 26 kg P and 90 kg K ha−1 y−1, as expected secondary limiting nutrients for plants) on microbial growth, soil C and N mineralization, microbial biomass, and community structure. Despite high levels of long-term fertilization, which significantly increased primary production, we observed relatively minor effects on soil microbial activity. Bacterial growth exhibited the most pronounced response to long-term fertilization, with higher rates of growth in fertilized soils, whereas fungal growth remained unaffected. Rates of basal soil C and N mineralization were only marginally higher in fertilized soils, whereas fertilization had no significant effect on microbial biomass or microbial community structure. Overall, these findings suggest that microbial responses to long-term fertilization in these subarctic tundra soils were driven by an increased flow of labile plant-derived C due to stimulated plant productivity, rather than by direct fertilization effects on the microbial community or changes in soil physiochemistry.