Dogs may play an important role on the emergence and dissemination of zoonotic parasitic diseases, including microsporidiosis. Microsporidia have emerged as important opportunistic protozoan parasites in immunocompromised individuals, such as HIV-positive patients. In this study, we performed a survey of occurrence of Microsporidia species in faecal and urine samples of dogs (n=250) from São Paulo city, Brazil. The samples were randomly selected from dogs housed in a university veterinary hospital (group I, n=150) and private kennels (group II, n=100). Urine sediment was obtained by centrifugation (1,000g for 20 minutes). Thin smears of stool and urine were screened for the presence of microsporidian spores by using Gram-Chromotrope. Furthermore, the presence of microsporidia was confirmed by their typical staining pattern with use of Weber´s modified Chromotrope-based stain. A smear was considered positive only if microsporidian spores were identified by both staining methods. It was observed 5 positive cases for microsporidia from faecal specimens, 3 from group I and 2 for group II. Using the polymerase chain reaction (PCR) technique, the spores were identified as Encephalitozoon cuniculi spores. No parasites were found in urine samples. By light microscopy, microsporidia spores were seen as purple and ovoid structures, in a green background, ranging from 2.4-3.2 micrometers of length to 1.0-1.6 micrometers of width. In this investigation, a prevalence of 2% for microsporidia was found in canine stools, showing that pets like dogs could be a potential source of these protozoans to humans.