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Soil microbial biomass and extracellular enzymes are involved in the decomposition of soil organic matter, a temporary nutrient pool for plants. This study was conducted to evaluate the mineralization potentials of carbon (CMP) and nitrogen (NMP), and the relationship of CMP and NMP with microbial biomass and extracellular enzymes in soil fertilized with mineral and manure amendments for 3 decades. A long-term experiment (37 years) consisted of seven fertilizer treatments, including unfertilized control (CK); nitrogen, phosphorus, and potassium (NPK); manure (M); manure plus phosphorus and potassium (PKM); nitrogen and potassium plus manure (NKM); nitrogen and phosphorus plus manure (NPM); and NPK plus manure (NPKM), was selected. Compared to the CK treatment, the long-term mineral fertilizer (NPK) and combined manure plus mineral fertilization (e.g., M, PKM, NKM, NPM, and NPKM) significantly increased soil microbial biomass carbon (SMBC) by 58% and 131% and nitrogen (SMBN) by 66% and 161%, respectively. Similarly, soil microbial biomass phosphorus (SMBP) also significantly increased by 67–156% in combined manure and mineral fertilizer treatments than the CK and NPK treatment. Higher values of β-glucosidase and cellobiohydrolase activities were observed with the application of PKM. In comparison with CK, the application of combined manure and mineral fertilization significantly increased the leucine aminopeptidase and N-acetyl-glucosaminidase enzyme by 129% and 175%, respectively. Soil CMP and NMP significantly increased by 231–307% and 176–289% in combined treatments than the CK. A significant positive correlation was also observed between the microbial biomass, extracellular enzymes, and the CMP and NMP (R2 = 0.66–0.84, 0.44–0.76, P