Enterocytozoon bieneusi and Giardia duodenalis are major zoonotic pathogens that often cause diarrhea in immunocompromised humans or animals. Beef cattle are important reservoirs for these two pathogens. Yunnan Province is a major region for beef cattle farming, and its suitable climatic conditions facilitate the transmission of the pathogens. However, research on the prevalence and distribution of E. bieneusi and G. duodenalis in beef cattle in Yunnan remains understudied. This study collected 529 fecal samples from seven beef cattle breeds in four regions in Yunnan Province for an epidemiological survey. Nested PCR combined with sequencing was used to detect E. bieneusi and G. duodenalis, and the sequencing results were analyzed to determine genotypes or assemblage types. Our results demonstrate that the prevalence rates of E. bieneusi and G. duodenalis were 3.0% (16/529) and 3.6% (19/529), respectively. The study identified four genotypes of E. bieneusi, including I (n = 8), J (n = 4), BEB8 (n = 3), and BEB4 (n = 1). Both assemblages E (n = 18) and A (n = 1) were identified among G. duodenalis-positive animals. Phylogenetic analysis revealed that the E. bieneusi genotypes detected in this study belong to Group 2. In conclusion, these findings indicate that although the overall prevalence is relatively low compared to other regions, the presence of zoonotic Group 2 genotypes and assemblage A highlights the potential risk of cross-species transmission. Moreover, the results provide foundational data to support the development of region-specific surveillance and control strategies for bovine giardiasis and microsporidiosis, and emphasize the importance of the One Health approach in managing parasitic infections in livestock populations.