The soil microbial biomass is largely responsible for the transformation of organic matter and the associated mineralization of important nutrients that regulate plant productivity. While the controls on soil microbial biomass and activity in temperate ecosystems have been well investigated, there is a paucity of such information from tropical rain forest ecosystems. Hence, we used a natural soil gradient (nutrient-poor oxisols and nutrient-rich mollisols) to investigate seasonal changes in the size and activity of the soil microbial community in two primary tropical rain forest sites in southwestern Costa Rica. In a nutrient-poor oxisol, microbial biomass ranged from 952 μg/g in the dry season (February) to 1967 μg/g in the wet season (June). The greater microbial biomass estimates corresponded to greater rates of microbial activity in the oxisol forest; however, the active fraction of the soil microbial biomass remained consistently low, regardless of soil C availability. In addition, while there was very little seasonal variability in microbial C:N ratios, we found extreme variations in seasonal microbial C:P ratios, with the highest microbial C:P ratio occurring when microbial biomass C was at a maximum. Our results suggest the importance of P availability in regulating soil microbial processes and that decomposition during the wet season (of litter accumulated in the previous dry season) may play an important role in driving seasonal changes in microbial biomass and activity.