Background and Aims <p>Long-term fertilization profoundly influences soil health and agricultural sustainability, but its effects on ecosystem multifunctionality (EMF) in fragile karst agroecosystems remain poorly understood. In the karst regions of southwest China, inappropriate tillage and fertilization measures have triggered severe land degradation issues. We therefore conducted a 12-year experiment based on a maize-soybean rotation system in southwest China’s karst areas to identify effective fertilization strategies applicable in fragile karst agroecosystems.</p> Methods <p>The experimental treatments comprised an unfertilized control (CK), mineral fertilizer (NPK) alone, and NPK combined with straw or cattle manure at low (LSNPK, LMNPK) and high (HSNPK, HMNPK) rates. We assessed the effect of constrasting fertilization strategies on crop productivity, soil organic carbon stability, microbial community structure, and EMF.</p> Results <p>All fertilization treatments increased combined maize and soybean productivity relative to CK, but their effects on soil health diverged markedly. Among the five fertilization treatments, HMNPK maximized the proportion of large macroaggregates (&gt; 2000&#xa0;µm) and the organic carbon concentration within them. HMNPK also elevated the chemical stability of soil organic carbon compared to CK and HSNPK. Phospholipid fatty acid and biomarker analyses revealed the highest microbial biomass and necromass carbon under HMNPK. Consequently, HMNPK yielded the highest EMF index, outperforming HSNPK and NPK.</p> Conclusions <p>A nutrient management regime with high-rate manure input is an effective strategy for enhancing karst agroecosystem multifunctionality. This approach sustains high crop productivity while simultaneously improving soil structure, SOC stability, and microbial community abundance, thereby benefiting long-term agricultural sustainability.</p> Graphical abstract <p></p>