Roads are an increasingly common feature of forest landscapes all over the world, and while information accumulates regarding the impacts of roads globally, there remains a paucity of information within tropical regions. Here we investigate the potential for biodiversity impacts from an unmarked road within a rainforest protected area in Western Amazonia. We focus on three key taxonomic groups; amphibians, butterflies and birds, each of which have been shown to be both sensitive and reliable indicators of forest disturbance. In total, 315 amphibians of 26 different species, 348 butterflies of 65 different species, 645 birds representing 77 different species were captured using mist netting and 877 bird records representing 79 different species were recorded using point counts. We provide evidence to show that the presence of a small unmarked road significantly altered levels of faunal species richness, diversity, relative abundance and community structure. This was true to a varying degree for all three taxa, up to and potentially beyond 350 m into the forest interior. Responses to the road were shown to be taxon specific. We found increasing proximity to the road had a negative effect on amphibian and understorey bird communities, whilst butterfly and overall diurnal bird communities responded positively. We show that the impact on biodiversity extends up to at least 32 % of the whole reserve area; a serious impact under any scenario. This work provides support for recently voiced calls to limit networks of unmarked roads in order to realistically and effectively preserve natural levels of tropical biodiversity.