The natural habitat of Paracoccidioides brasiliensis remains undefined but the repeated demonstration of infection by this fungus in the nine-banded armadillo Dasypus novemcinctus has opened interesting research avenues. We report here the isolation of this fungus from the spleen of a naked-tailed armadillo Cabassous centralis (Miller 1899) captured in a coffee farm localized in the Colombian endemic area for paracoccidioidomycosis. This particular isolate was identified by its dimorphism and also by comparison of the PbGP43 gene and ribosomal internal transcribed spacer regions (ITS) with recognized P brasiliensis strains. This finding extends the range of naturally acquired infections in mammals of the family Dasypodidae and confirms the existence of this human pathogen in areas where human paracoccidioidomycosis is known to occur.