As a part of the developing bioeconomy, liquid biofuels may play an important role for transportation due to the hope for a sustainable drop-in alternative to substitute fossil fuels and maintaining existing economic infrastructures. In this case study we applied Holistic and Integrated Life Cycle Sustainability Assessment to a prospective technical concept for the production of biofuels from wood residues, sorghum and straw via gasification and Fischer-Tropsch synthesis located in the German federal state Brandenburg. Through this quantitative and qualitative sustainability assessment we identified synergies and hot-spots of biofuel production on a detailed and aggregated level, as well as compare the impacts to fossil fuels and other alternative transport systems. 99 social, ecological and economic indicator results addressing 14 out of 17 SDGs show contributions but also sustainability risks of such biofuels for the SDGs. The total substitution factor of impacts (f = 21.38) for all indicators of biofuels compared to fossil fuels indicates significantly higher impacts of biofuels production, in particular for land (SDG 15, f = 30.43), water (SDG 14, f = 125.57), consumption and production patterns (SDG 12, f = 54.11), low energy efficiency and maintaining problematic global supply chains and working conditions. However, the impacts on climate can be lower (SDG 13, f = 0.42), if residual heat is effectively and efficiently used. Comparing the transportation systems and use phases of fuels, all types of car-based individual transportation including fossil fuels (f = 6.50), biofuels (f = 9.16) and electric drive (f = 6.46) had significant higher impacts than transportation by train. Besides technological downsides, such as the high energy demand, biofuels may play a minor role for specific applications with no other alternative energy technologies in the future. In conclusion, it is very questionable whether such liquid biofuels are a suitable drop-in solution to substitute fossil fuels in a significant quantity. In a final discussion we referred to the necessary societal-ecological transformation with structural changes of production, consumption, political economy and global supply chains. In the future, Holistic and Integrated Life Cycle Sustainability Assessment will be further improved by closing indicator and database gaps, including a cost analysis and direct stakeholder participation, as well as absolute sustainability assessments on how much biofuel production is sustainable.