This study was carried out in the Private Natural Heritage Reserves Lote Cristalino (670 ha), Cristalino (6,539 ha) and in Cristalino State Park (184,900 ha). These protected areas are contiguous and are located in the extreme north-central State of Mato Grosso, in areas considered priorities for conservation due to the high biodiversity and endemism and the high human pressure. Thus, this study focused to evaluate and compare the structure of populations of medium and large mammals in terms of richness and abundance in environments without tourism and with ecotourism activities in Cristalino region. Therefore, in the period from May 2008 to February 2010, primary forest environments with two levels of anthropogenic disturbance were sampled: no tourism at all and ecotourism activities. Data were collected by using the distance samplings in linear transect, totalizing 468,3 kilometers traveled during daytime and nighttime, and the record of footprints in previously prepared plots (n = 660 surveyed plots), in addition to river corridors in Cristalino River and random searches in places with difficult access due to the lack of roads. Records of 37 species of mammals were obtained, which 33 were medium and large size and four small ones. There was no statistically significant difference in species richness of the environments without tourism and with ecotourism, and the similarity of species among them was quite high (88%). However, three taxa were less abundant in environments with tourism: Dasyprocta leporina (Linnaeus, 1766), Mazama spp. and Dasypus kappleri Krauss, 1862. Therefore, it can be observed that the negative impact of ecotourism activities developed in the study area presented a small magnitude, in terms of richness and abundance of medium and large mammals. Thus, ecotourism ventures are presented as important economic activity to be developed in areas with touristic potential in the Amazon.