Soil carbon (C) pools and biological indicator plays an important role in maintaining soil quality. Land use change from naturally undisturbed to intense cultivation had significant impact on C storage due to change in soil biological properties. The present study was conducted to understand the impact of land use change from the uncultivated to intensively cultivated soils under rice–wheat and cotton–wheat cropping system on labile and stable C pools, soil microbial and enzymatic activity, and a change in nutrient availability. The Olsen’s P concentration was significantly (p < 0.05) higher in soils under rice–wheat, while ammonium acetate (NH₄OAc)-K was higher in the uncultivated soils, compared with others. Soils under cotton–wheat cropping system had significantly lower total organic carbon (TOC) in contrast to rice–wheat soils. The uncultivated soils had the highest TOC concentration, which was ∼20.2% higher than rice–wheat and ∼46.4% than the cotton–wheat soils. The water extractable organic carbon (WEOC) was the smallest organic C fraction (∼0.31%–0.40% of TOC), and was significantly lower in cotton–wheat soils. The basal soil respiration (BSR) and mineralization quotient (qM) were significantly higher for rice–wheat, compared with the cotton–wheat soils. These microbial indices were improved due to increased alkaline phosphates (Alk-P) and dehydrogenase (DHA) enzymatic activity in soil. Soil microbial biomass C (MBC) was significantly higher (by ∼31.7%–57.3%) in the uncultivated than the cultivated soils which has increased the C mineralization in soil. The stable C pool comprised ∼68.8% of TOC in the uncultivated soils, almost similar to rice–wheat soils (∼68.5%), but higher than cotton–wheat soils (∼61.9%). The stable C pool leads to C rehabilitation indicated by higher C management index for rice–wheat soils. The principal component analysis (PCA) distinct the uncultivated soils from the cultivated soils under two cropping systems based on BSR and labile C (Fract. 2) in PC₁ (explaining ∼82.7% of total variability), and the microbial (qmic) and mineralization (qM) quotient in PC₂ (∼17.3% variability). These indicators were most influential for studying C dynamics of the natural ecosystem and restoring the cropland ecosystem by suitable soil management practices.