As the current therapies for intestinal microsporidiosis are either inconsistent in their efficacies or hampered by several adverse effects, alternative antimicrosporidial agents are being sought. The present study is the first that was designed to evaluate the potency of orlistat, an approved anti-obesity drug, against intestinal microsporidiosis caused by both Enterocytozoon bieneusi and Encephalitozoon intestinalis. Results were assessed through studying fecal and intestinal spore load, intestinal histopathological changes, viability, and infectivity of spores from treated animals. Results showed that orlistat has promising antimicrosporidia potential, with better results in E. intestinalis than E. bieneusi. The animals that received orlistat showed statistically significant decrease in the fecal and intestinal spore load, when compared to the corresponding control infected nontreated mice. The results were insignificant compared to fumagillin and albendazole. Light microscopic examination of stained intestinal sections revealed amelioration of the pathological changes and decreased inflammatory cells detected in the control infected nontreated mice. Spores encountered from stool of orlistat-treated E. bieneusi and E. intestinalis mice showed low viability and significant reduction of infectivity versus their control. Thus, considering the results of the present work, orlistat proved its effectiveness against the intestinal microsporidial infection.