The present paper reports the occurrence of natural co-infection of Babesia caballi and Encephalitozoon-like microsporidia in the tick Anocentor nitens. Engorged females of ticks, collected from a naturally B. caballi-infected horse, were incubated at 27 degrees C and relative humidity over 83%. After a 6-day incubation period, Giemsa-stained smears prepared from hemolymph were examined microscopically under oil immersion. B. caballi infected ticks were dissected and samples of midgut tissue were examined by transmission electron microscopy, through which free sporokinetes were seen in the cytoplasm of gut epithelial cells. In addition, Encephalitozoon-like microsporidia were observed inside the parasitophorous vacuoles in the same cell in which sporokinetes of B. caballi were found and also in some neighbour cells. They presented different morphological stages, suggesting a sequential phases of development.