Today, Brazil is the second major soybean (Glycine max (L.) producer of the world; however, few studies on the effects of transgenesis in the soil microbiota are completed. This study aims at assessing the residual effect of a transgenic soybean with tolerance to the imidazolinone herbicide group on the soil microbiota. The parameters examined were: the soil basal respiration, microbial biomass carbon (MBC) of the soil, metabolic quotient, spores of arbuscular mycorrhizal fungi and total soil fungal and bacterial biomass using epifluorescence technique in areas with eight years of no-tillage cultivated with non-transgenic (BRS133) and transgenic (BRS245) soybeans, in Londrina and Ponta Grossa -PR, Brazil, in the 2011/2012 growing season. The experiment was conducted in a randomized block design and each treatment with six replications. Results showed a significant increase on the MBC in Londrina soils with the BRS245 soybean, and also a significant increase in the total bacterial biomass in the both Londrina and the Ponta Grossa soil with BRS245 soybean, in contrast to those cultivated with BRS133. The BRS245 transgenic soybean crop changed the soil microbiota mainly by increasing the bacterial biomass, which was estimated by the epifluorescence microscopy method. The epifluorescence technique can be used to estimate soil biomass by fungi and bacteria with success.