BACKGROUND: Intestinal microsporidiosis is an emerging opportunistic infection that primarily affects individuals with compromised immune systems. This study investigated intestinal microsporidia infections in individuals diagnosed with multiple sclerosis (MS) and elucidated the genetic diversity and evolutionary relationships of microsporidia. METHODS: A total of 116 stool samples were collected from individuals diagnosed with MS, including 54 men and 63 women, during 2022-2023 in Kurdistan Province, western Iran. The mean age of the participants was 38.28 ± 7.8 years. RNA extraction was performed, followed by amplification of the small subunit ribosomal RNA (SSU rRNA) to identify and characterize microsporidia and their associated genetic markers. A phylogenetic tree was constructed using MEGA X software to explore the evolutionary relationships among the isolates. RESULTS: PCR revealed Enterocytozoon bieneusi in 5 of 116 samples (4.3 %), whereas no positive cases of Encephalitozoon species were detected. Additionally, no statistically significant associations were observed between the presence of microsporidia and variables such as age, sex, or geographic region. CONCLUSION: This study highlights the clinical significance of Enterocytozoon bieneusi in immunocompromised populations, particularly in patients with multiple sclerosis (MS). The findings underscore the potential role of zoonotic transmission and highlight the urgent need for enhanced diagnostic capabilities and preventive strategies to combat intestinal microsporidiosis in vulnerable groups, particularly in regions such as western Iran.