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The present study was aimed at establishing microbial activity and diversity in two palaeosols (P1 and P2) intercalated within the late Quaternary continental sequences of Mahi River basin, western India. The two palaeosols have been dated back to similar to 45 and 30 ka. High microbial biomass carbon, soil respiration, microbial quotient, metabolic quotient and dehydrogenase activity revealed that the organic matter decomposition and carbon flow to the atmosphere were much higher in P1. Soil in situ enzymes, specific enzymatic quotients and specific enzyme indices revealed that P1 has been more productive than P2. The order of biogeochemical cycling in P1 was N > P >= C, whereas P2 showed N > P > C. 16S rRNA gene profiling using denaturing gradient gel electrophoresis indicated greater heterotrophic bacterial diversity and higher carrying capacity of P1 compared to P2. The contrasting microbial activities and diversity in these palaeosols point to different environments during palaeosol formation, and are in general agreement with the climatic inferences drawn earlier.