In this study, we analyze the impact of fertilizer- and manure-induced N2O emissions due to energy crop production on the reduction of greenhouse gas (GHG) emissions when conventional transportation fuels are replaced by first-generation biofuels (also taking account of other GHG emissions during the entire life cycle). We calculate the nitrous oxide (N2O) emissions by applying a statistical model that uses spatial data on climate and soil. For the land use that is assumed to be replaced by energy crop production (the ‘reference land-use system’), we explore a variety of options, the most important of which are cropland for food production, grassland, and natural vegetation. Calculations are also done in the case that emissions due to energy crop production are fully additional and thus no reference is considered. The results are combined with data on other emissions due to biofuels production that are derived from existing studies, resulting in total GHG emission reduction potentials for major biofuels compared with conventional fuels. The results show that N2O emissions can have an important impact on the overall GHG balance of biofuels, though there are large uncertainties. The most important ones are those in the statistical model and the GHG emissions not related to land use. Ethanol produced from sugar cane and sugar beet are relatively robust GHG savers: these biofuels change the GHG emissions by −103% to −60% (sugar cane) and −58% to −17% (sugar beet), compared with conventional transportation fuels and depending on the reference land-use system that is considered. The use of diesel from palm fruit also results in a relatively constant and substantial change of the GHG emissions by −75% to −39%. For corn and wheat ethanol, the figures are −38% to 11% and −107% to 53%, respectively. Rapeseed diesel changes the GHG emissions by −81% to 72% and soybean diesel by −111% to 44%. Optimized crop management, which involves the use of state-of-the-art agricultural technologies combined with an optimized fertilization regime and the use of nitrification inhibitors, can reduce N2O emissions substantially and change the GHG emissions by up to −135 percent points (pp) compared with conventional management. However, the uncertainties in the statistical N2O emission model and in the data on non-land-use GHG emissions due to biofuels production are large; they can change the GHG emission reduction by between −152 and 87 pp.