Soil enzyme activities provide unique biochemical assessment of soil function as a good indicator of soil fertility which can be altered due to the profusion of fluoride in soil and seasonal change. Seven sites were chosen in the fluoride affected area of Nasipur, Birbhum district, West Bengal, India, to make a comparative assessment of the seasonal changes of enzyme (urease, amylase, cellulase and invertase), fluoride content, physicochemical characteristics along with the availability of microbes in the soil with control. Results revealed that all the enzymes show variable activity with season. However urease showed higher activity during summer followed by winter. But urease activity showed marginal differences with control area during winter (p < 0.002) and summer (p < 0.110) with significant (p < 0.000) difference during rainy season. Soil pH showed negative impact on urease during winter and summer. Cellulase was found to be accelerated by both organic matter and organic carbon content in the soil. An influence of fluoride activity during rainy, summer and winter season was noticed in case of urease amongst the studied enzymes. Microbial population in the soil demonstrated negative impact of fluoride on it which in turn may affect the soil enzyme and character as well.