Roads are essential for social and economic development, but their presence often leads to deforestation, habitat loss, changes in wildlife behaviour and distribution, and loss through collisions with vehicles. In southern Bahia state in Brazil’s Atlantic Forest hotspot, cocoa agroforestry systems are vital in connecting natural fragments and supporting local biodiversity. This study assessed the impacts of a 24 km segment of the BA-262 road, a route linked to the construction of a new port. During May–December 2022, we recorded vertebrate roadkill events and analysed their relationship with traffic volume, identifying high-risk zones. Camera traps and live traps were deployed in forest fragments to evaluate mammal presence near and along the road, comparing species detected near the road with those recorded as roadkill. In total, we documented 243 roadkill events, with amphibians being the group most affected (153 individuals, 63% of the total recorded), followed by reptiles (49, 20%), birds (28, 12%) and mammals (13, 5%). Hotspot analyses revealed nine roadkill clusters. Camera traps along the road recorded 409 images of 21 mammal species from eight orders. Although species richness was unaffected by proximity to the road, relative abundance increased closer to the road. Mammals appeared less affected by the road than other groups, probably because they are mostly active at night, when traffic is lower. This research highlights the ecological impacts of roads on biodiversity-rich areas, underscoring the need for mitigation measures to reduce wildlife mortality and offering insights for future research.