The global demand for biofuels in the transport sector may lead to significant biodiversity impacts via multiple human pressures. Biodiversity assessments of biofuels, however, seldom simultaneously address several impact pathways, which can lead to biased comparisons with fossil fuels. The goal of the present study was to quantify the direct influence of habitat loss, water consumption and greenhouse gas (GHG) emissions on potential global species richness loss due to the current production of first‐generation biodiesel from soybean and rapeseed and bioethanol from sugarcane and corn. We found that the global relative species loss due to biofuel production exceeded that of fossil petrol and diesel production in more than 90% of the locations considered. Habitat loss was the dominating stressor with Chinese corn, Brazilian soybean and Brazilian sugarcane having a particularly large biodiversity impact. Spatial variation within countries was high, with 90th percentiles differing by a factor of 9 to 22 between locations. We conclude that displacing fossil fuels with first‐generation biofuels will likely negatively affect global biodiversity, no matter which feedstock is used or where it is produced. Environmental policy may therefore focus on the introduction of other renewable options in the transport sector.