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<i>Enterocytozoon bieneusi</i>, an intracellular eukaryote closely related to fungi, is recognized as a significant pathogen affecting humans, particularly those with compromised immune systems. While its transmission routes are still not fully elucidated, fecal–oral transmission remains the primary one. With a wide host range, the zoonotic potential of <i>E. bieneusi</i> is a concern, albeit direct evidence of animal-to-human transmission remains scarce. Genotyping based on the internal transcribed spacer (ITS) region facilitates the delineation of genetic diversity, with potentially zoonotic genotypes predominantly associated with Groups 1 and 2. Despite the broad spectrum of susceptible animal hosts, research into microsporidian infection among zoo animals remains limited. This study aimed to evaluate the occurrence of <i>E. bieneusi</i> infection across diverse captive animals, focusing on zoo settings in Portugal. Fecal samples were collected from a variety of animals, and molecular detection of <i>E. bieneusi</i> was conducted using nested PCR targeting the ITS region. Of 127 fecal samples, 1.57% (95% CI: 0.19–5.57) tested positive for <i>E. bieneusi</i>, with non-human primates (NHP’s) exhibiting an 18.18% (95% CI: 2.28–51.78) occurrence. Phylogenetic analysis revealed clustering within Group 2 genotypes, indicating potential zoonotic implications. This study highlights the need for further research to understand the epidemiology of <i>E. bieneusi</i> in zoo environments and its potential transmission pathways to humans.