Background: Microsporidiosis is considered as an opportunistic infection in immunodeficient patients. Objectives: Due to the increasing prevalence of parasitic infections and immunodeficiency diseases as well as the transmission risk of microsporidia from animals to human, the aim of this study was to evaluate molecular diagnosis of Enterocytozoon bieneusi and Encephalitozoon spp. by the multiplex/nested polymerase chain reaction (PCR) and staining methods in wild rats of Ahvaz city, southwest of Iran. Methods: Initially, 160 stool samples were collected from wild rats in different parts of Ahvaz city. The samples were stained by the modified trichrome staining and explored microscopically. The DNA was extracted using the DNA stool kit and examined by multiplex/nested PCR. For differentiating the species of Encephalitozoon, the multiplex/nested PCR products were explored by the restriction fragment length polymorphism (RFLP) method using the restriction enzyme of Mnl1. Results: Of 160, 26 cases were suspected positive for microsporidia spore by the staining. Also, of 160, 18 cases were positive by the multiplex/nested PCR method, that 14 and 4 samples were detected as E. bieneusi and E. intestinalis, respectively. Of 14 E. bieneusi samples, 12 and 2 cases were detected as genotype D and M, respectively. Conclusions: The findings revealed a relatively high prevalence of microsporidia infection in wild rats of the city and these animals can be a source of microsporidiosis. Due to the zoonotic potential of the microorganisms, high -risk individuals should be receiving the information about the risk of direct and indirect contact with the infected animals.