Abstract Due to growing interest in biofuels as alternative renewable energy sources, several recent studies have assessed the sustainability of their production. Emergy is a widely used environmental indicator for this purpose, as it counts exploitation of natural resources and direct and indirect solar energy requirements of biofuel production. Depending on whether a biofuel is first, second or third generation, its production system differs in nature and the indications derived from emergy evaluations vary as well. This article aims to provide guidelines on how to interpret and properly use the results of emergy evaluation of first, second and third generation biofuels. These guidelines are useful for correct emergy assessment of biofuels and clarify the actual meaning of emergy evaluation outcomes.