In 2013, we conducted a baseline study on the presence, distribution and occupancy of medium- to large-sized mammals in Bolivia along the San Buenaventura-Ixiamas road that runs parallel to the Madidi National Park and Natural Area of Integrated Management boundary and cuts through the Tacana Indigenous Territory and a number of neighboring private properties. Establishing a 3 km buffer on each side of the road, we studied an 865 km2 area divided into 1 km2 cells and sampled a total of 356 of these cells. In each cell, we established one 300 m transect divided into 25 m sections and registered wildlife sign, mainly footprints, from eight wildlife species or species groups. The transects were placed either along streams (75% of cells) or within forest (25% of cells). Using single-season single-species occupancy models we estimated occupancy (ψ) for Tapirus terrestris (ψ = 0.39), Pecari tajacu (ψ = 0.5), Mazama americana (ψ = 0.56), Dasyprocta spp. (ψ = 0.59), Cuniculus paca (ψ = 0.56), Leopardus spp. (ψ = 0.33), and use for Tayassu pecari (ψ = 0.17) and Panthera onca (ψ = 0.11). Occupancy and use results verified community perceived wildlife corridors between Madidi and its area of influence. We identified additional corridors along many streams crossing the San Buenaventura-Ixiamas road. This connectivity is not only important for wildlife, but also from a food security perspective for the indigenous communities that depend on wildlife as an important source of protein. The results will be used to mitigate the impact of road improvements through the identification of priority areas for maintaining connectivity between Madidi and the surrounding landscape.