In the present study, we aimed to characterize and quantify the vertebrates road-killed along 12.8 km of the BR-330 highway in southwestern Bahia. The road is surrounded by patches of Atlantic Forest and Caatinga and by human-modified areas. From May/2012 to August/2013, we performed 35 surveys, monitoring 448 km in total. During this period, we registered 146 road-killed individuals belonging to 60 species (35 birds, 15 reptiles, 7 mammals and 3 amphibians). The species most likely to be road-killed in the study area were the toad Rhinella jimi and the snakes Oxyrhopus trigeminus and Sibynomorphus neuwiedi . The Jaccard index showed higher values when compared to the observed richness of road-killed species, which is corroborated by the ten new species recorded during five additional surveys from August/2015 to July/2016. The number of road-killed vertebrates was significantly higher in the rainy season than in the dry season. We identified 26 road-kill hotspots along the studied stretch of road. The presence of water bodies associated with vegetational landscape surrounding these hotspots may be a reason for the higher concentration of road-kills. Besides being one of the few studies conducted in an ecotonal area in the northeast of Brazil, our data indicate some important patterns that should be considered for regional and national policies on impact mitigation.