Purpose <p>Vegetation disturbance significantly impacts soil nitrogen cycling and microbial communities. Nonetheless, our comprehension of how varying intensities of vegetation disturbance impact free-living nitrogen fixation (FLNF) rates remains limited. This research examines the influence of different magnitudes of vegetation disturbance (slight, moderate, and extreme) in a karst region on soil free-living nitrogen fixation (FLNF) rates.</p> Methods <p>The investigation considers how these disturbances modify soil physicochemical attributes and affect the abundance, diversity, and interactions of diazotrophs and arbuscular mycorrhizal fungi (AMF).</p> Results <p>Our findings indicate that areas experiencing moderate to severe vegetation disturbance exhibited a marked reduction in soil FLNF rates, key nutrient concentrations (including total phosphorus, nitrate, available phosphorus, and total potassium), and soil moisture, when compared to sites undergoing natural vegetation restoration, while concurrently showing an increase in soil pH. Moreover, increasing vegetation disturbance significantly reduced diazotroph abundance, whereas, unexpectedly, both the abundance and diversity of AMF increased. Network analyses further revealed stronger interactions among AMF taxa under high vegetation disturbance, whereas the cooperative relationships between diazotrophs and AMF were weakened. Under strong vegetation disturbance, the combined effects of reduced nitrate and phosphorus availability, lower soil moisture, and weakened diazotroph–AMF interactions contributed to the suppression of FLNF rates.</p> Conclusion <p>Our findings underscore the critical need for sustainable vegetation management to preserve soil microbial functionality and nitrogen cycle stability in fragile karst ecosystems.</p>