This study investigates the presence of Enterocytozoon bieneusi in domestic and wild animals living in the wildlife/livestock interface area of the Kruger National Park (KNP) in South Africa. Fifty fecal samples from domestic calves in rural communities and 142 fecal samples from impala (Aepyceros melampus) and buffalo (Syncerus caffer) in the KNP were analysed for Enterocytozoon bieneusi, using a nested PCR targeting the internal transcribed spacer of the rRNA gene. All wildlife samples were negative for E. bieneusi, whereas nine (18%) calf samples were positive. Three cattle specific genotypes (group 2) were identified, belonging to the known genotypes BEB4 and I, and one novel genotype (BEB3-like). One human-pathogenic genotype (D) was detected in one calf. This is the first study on microsporidia performed in a wildlife/livestock interface area of sub-Saharan Africa. Our findings show that at least one genotype of zoonotic importance is circulating in native cattle in the study area and the rest of the identified microsporidia were host-specific genotypes. Larger studies in domestic animals, humans and wildlife are necessary to assess the public health significance of E. bieneusi in that interface area.