The microbial biomass carbon (MBC) and carbon mineralization of fly ash (FA) amended soil at (0 %, 1.25 %, 2.5 %, 5 %, 10 % and 20 % FA; v/v) was investigated under laboratory conditions for 120 days at 60 % soil water-holding capacity and 25 ± 1°C temperature. The results demonstrated that soil respiration and microbial activities were not suppressed up to 2.5 % FA amendment and these activities decreased significantly at 10 % and 20 % FA treatment with respect to control. Application of 10 % and 20 % FA treated soils showed a decreasing trend of soil MBC with time; and the decrease was significant throughout the period of incubation. The study concluded that application of FA up to 2.5 % can thus be safely used without affecting the soil biological activity and thereby improve nutrient cycling in agricultural soils.