The conversion of tropical forests to monoculture rubber plantations throughout Southeast Asia threatens to have widespread negative impacts on ecosystem services. The aim of this study was to identify the impacts of forest conversion to rubber plantations on soil microorganisms, using a space for time substitution design. Soil microbial communities from natural forests, young rubber and mature rubber plantations were investigated using phospholipid fatty acid (PLFA) analysis. Microbial PLFA biomass of all functional groups including actinomycete, arbuscular mycorrhiza, fungi, bacteria and protozoa was lower in rubber plantations as compared to natural forests. This was true for young rubber and mature rubber plantations tested in both dry and wet seasons. The composition of microbial communities differed between natural forests, young rubber and mature rubber plantations. Changes in microbial community composition were strongly correlated with pH, which was lower in mature rubber plantations than in natural forests and young rubber plantations. In addition, our results showed that the variation in soil microbial community composition was strongly affected by seasonality, with higher PLFA biomass recorded in the dry season than in the wet season. The low soil microbial biomass in rubber plantations has long term implications regarding the soil health of rubber plantations.