Road infrastructure is expected to expand globally in the coming decades. The infrastructure construction is expected to be particularly intense in the Global South, where most of the world’s biodiversity is concentrated. Current data on road effects on the environment predominantly comes from studies on certain species. This may signal potential underestimation of the environmental impacts, which is likely to result in application of insufficient mitigation measures, ultimately undermining biodiversity conservation efforts. This can pose a global threat to provision of vital ecosystem services, ecological integrity and human wellbeing. Ultimately, the damage caused by road infrastructure development may outweigh economical benefits. Thus, lack of understanding of potential consequences of road infrastructure development is very dangerous and needs to be urgently addressed. In the present article the potential impacts of road infrastructure on biodiversity are reviewed. Based on this review effectiveness of currently widespread mitigation measures is critically evaluated and recommendations to policymakers and conservation practitioners are provided.