Mining activities lead to land degradation and alter ecosystem functions. Monitoring land degradation status is essential to take appropriate and timely conservation measures. Soil genesis during early years of mine spoil reclamation is critical and may help to predict reclamation success. The microbial activity is significantly influenced by the physicochemical properties, and hence, the assessment of these changes is essential for soil management practices. In the present investigation, the physico-chemical characterization and the activities of six different enzymes (amylase, invertase, protease, urease, phosphatase and dehydrogenase) were periodically analyzed with respect to different coal mine overburden spoil in chronosequence over a period of 10 yr, and compared with the native forest soil, in order to assess their effectiveness in reclaiming mine overburden spoil. Comparative analysis suggested that there was gradual increase in enzyme activities from a nutrient deficient situation (fresh mine spoil) to an enriched soil (native forest soil). Besides, the variation in enzyme activities was significantly attributable to differences in physico-chemical properties. Stepwise multiple regression analysis was performed in order to determine the contribution of different physico-chemical properties influencing the variability in enzyme activities. Further, principal component analysis was able to discriminate six coal mine overburden spoils and native forest soil into independent clusters on the basis of their physico-chemical properties and enzyme activities. The study clearly revealed that the change in microbial indices in terms of enzyme activities were more responsive and correlated very well with the extent of land degradation, and therefore, can serve as biomarker for reclamation studies.