Background Intestinal microsporidiosis is an opportunistic infection associated with persistent diarrhea among HIV/AIDS patients. In Yemen, however, its epidemiology is unknown. Therefore, this study determined its prevalence and predictors among HIV/AIDS patients receiving antiretroviral therapy (ART) in Sana’a city, the capital of Yemen. Methods This cross-sectional study included 402 patients receiving ART at Al-Jomhori Educational Hospital in Sana’a from November 2019 to December 2020. Data about demographics, clinical characteristics and risk factors were collected using a pre-designed questionnaire. Stool samples were collected and examined for microsporidian spores using the Gram-chromotrope Kinyoun staining. Blood samples were also collected and used for CD4 cell counting by flow cytometry. Univariate analysis was used to test the association of patients’ characteristics and risk factors with intestinal microsporidiosis. Multivariable logistic regression was then used to identify the independent predictors of infection. Statistical significance was considered at P-values < 0.05. Results Intestinal microsporidiosis was prevalent among 14.2% (57/402) of HIV/AIDS patients and was significantly associated with diarrhea (OR 3.4, 95% CI 1.7–6.6; P = 0.001). The significant independent predictors of infection were < 200 CD4 cells/µl (AOR 3.2, 95% CI 1.5–6.9; P = 0.003), not washing hands after contacting soil (AOR 2.5, 95% CI 1.1–5.4; P = 0.026) and before eating (AOR 3.1, 95% CI 1.5–6.4; P = 0.003), eating unwashed raw produce (AOR 2.5, 95% CI 1.2–5.3; P = 0.017) and absence of indoor latrines (AOR 6.2, 95% CI 1.5–25.9; P = 0.012). Conclusions The prevalence of intestinal microsporidiosis among HIV/AIDS patients in Sana'a is high and comparable to that reported from several other countries, being prevalent among approximately 14.0% of patients and significantly associated with diarrhea. It could be predicted among patients who have < 200 CD4 cells/µl, have poor hand hygiene after contacting soil and before eating, usually eat unwashed raw produce, or do not possess indoor latrines. Large-scale studies on its epidemiology and predictors among HIV/AIDS patients across the country are warranted.