Aims <p>Nitrogen (N) fixation by moss-cyanobacteria associations contributes significant N to N-deficient ecosystems but remains unstudied in degraded karst landscapes with severe N limitations. We aimed to determine how host moss identity and microclimate regulate N₂-fixation and associated cyanobacterial biomass in degraded karst ecosystems, addressing a key knowledge gap for sustaining N input in these highly vulnerable habitats.</p> Methods <p>Associated cyanobacterial composition, biomass&#xa0;(phycocyanin), and nitrogenase activity (acetylene reduction assay) were quantified for four dominant epilithic moss species across five ecological degradation levels and three elevational gradients. Additionally, we explored and compared the key drivers (including microclimate, moss traits, and host identity) influencing these two variables.</p> Results <p>Our results indicated that N₂-fixation rates are peaking in low-elevation transect and light rocky desertification. Cyanobacterial biomass of moss-associated was not correlated with nitrogenase activity. Host moss species play the most important role in determining the associated cyanobacterial biomass and nitrogenase activity in degraded karst ecosystems. Furthermore, N, maximum water holding capacity and phosphorus were identified as the primary positive drivers of cyanobacterial biomass and molybdenum and phosphorus were identified as the primary positive drivers of nitrogenase activity. Microclimate significantly modulated activity: UV-A and light intensity were negative drivers, while air temperature had stronger positive effects than humidity.</p> Conclusions <p>Host moss species identity is the primary regulator of N₂-fixation in degraded karst ecosystems, mediated by species-specific traits (nutrient stoichiometry). This reveals a significant, previously overlooked biological N source critical for sustaining N input in these vulnerable habitats.</p>