Enterocytozoon bieneusi is a fungus-like protist parasite that can cause diarrhea and enteric diseases. The infection of E. bieneusi has been reported in many host species, including cattle and humans. However, information on prevalence and genotype distribution of E. bieneusi in dairy cattle in Yunnan province in China is still absent. In this study, 490 Holstein Cows and 351 dairy buffalo fecal samples were collected from three regions in Yunnan province, China. By using nest-PCR that targets the internal transcribed spacer (ITS), we found that the prevalence of E. bieneusi was 0.59% (5/841). DNA sequence analysis showed that five E. bieneusi genotypes were identified in this study, including two novel genotypes, YNDCEB-90 and YNDCEB-174, and three known genotypes (I, J, BEB4). Phylogenetic analysis revealed that two novel genotypes, YNDCEB-90 and YNDCEB-174, were clustered into Group 1, representing the zoonotic potential. The remaining genotypes I, J, and BEB4, which are the most frequent genotypes of E. bieneusi infection in cattle and lead to E. bieneusi infection in humans, belonged to Group 2. Although the lower prevalence of E. bieneusi was detected in dairy cattle in Yunnan province, it indicates that dairy cattle should be considered to be one of the potential hosts for transmitting E. bieneusi to humans. These findings are important for the development of effective prevention strategies for microsporidiosis.