Understanding the effects of long-term use of fertilizers on soil carbon and nitrogen pools and their activities is essential for sustaining soil productivity. Our objectives were to quantify long-term changes in soil organic carbon (SOC), soil microbial biomass carbon (SMBC), soil microbial biomass nitrogen (SMBN) and mineralizable C in maize–wheat cropping sequence in fertilized and unfertilized plots (control, N, NP, NPK, and NPK + FYM). Continuous application of fertilizers increased SOC over its initial content. Active fractions of SOC, i.e., water-soluble carbon, hydrolysable carbohydrates, SMBC, SMBN and dehydrogenase activity, improved significantly with an application of NPK and NPK + FYM. A general increase in carbon mineralization with time period was observed throughout the experiment and was maximum in 100% NPK + FYM treated plots. The estimated annual C input value in NPK + FYM treatment was 1.05 MgC ha−1 year−1. The overall net change in organic carbon was maximum in treatment receiving FYM along with inorganic fertilizers. Therefore, these results suggest that the integrated use of NPK and FYM is an important nutrient management option for sustaining maize–wheat cropping system.