High rates of paracoccidioidomycosis disease and infection have been recently reported in southern Brazil. However, the knowledgment about the ecoepidemiology of Paracoccidioides spp. in the region is limited, therefore, this study aimed to evaluate the presence of Paracoccidioides spp. DNA in soil samples from a rural area in Southern Rio Grande do Sul, Brazil. Thirty pools of soil samples from Bagé, RS (31º19'53"S 54º06'25"W) were submitted to physicochemical analysis, and to fungal DNA extraction by Norgen Biotek® Kit (Thorold, Canada), followed by Nested PCR technique with ITS4 and ITS5 as external primers, and PBITS-E and PBITS-T as internal primers. DNA amplification products of about 424 bp compatible with Paracoccidioides spp. were detected in eight (26.7%) of the 30 pools of samples, being three were sequenced and identified as P. brasiliensis. Positive soils were characterized by high levels of humidity, organic matter, basic saturation, and pH. This study shows for the first time the presence of Paracoccidioides spp. DNA at soils from the Brazilian Pampa Biome, proving that people living in those areas are exposed to the main agent of paracoccidioidomycosis.