ABSTRACT The different physiognomies and soil conditions across the Cerrado gradient may influence soil microbial biomass. The present study evaluated the soil microbial biomass and enzyme activity across a preserved Cerrado gradient and correlated these with environmental conditions. The site, sampling period and their interaction influenced soil microbial biomass and activity. Soil conditions, i.e., chemical and microclimatic properties, varied across the Cerrado gradient and influenced soil microbial biomass and activity. The highest and lowest values for microbial biomass and enzyme activity were found in Cerradao and Campo graminoide, respectively, during both seasons. Multivariate analysis showed that the sites were clearly separated into different groups, indicating that distinct physiognomies and environmental conditions influenced soil microbial biomass and enzyme activities.