World is heading towards the crisis of fossil fuel. Energy crisis is more acute in Bangladesh, as there is no petro-fuel source but only natural gas. It has among the lowest per capita energy (240 kg oil equivalents) consumption in the world and is severely dependent on additional environmentally friendly renewable energy resources in the future. To cope up with present situation and to reduce dependency on imported fuel, Bangladesh government is encouraging the use of renewable energy sources. In this circumstances, biofuel can be a very good alternative fuel for transportation. This paper gives insight into biofuel production feasibility and its usage for road transport can play an important role in the biggest metropolitan city of Bangladesh and contributes to knowledge on how to perform similar studies. Resource-focused assessment including feedstock from the waste sector, agricultural sector, forestry sector and aquatic environments partially considering technological and economic constraints. Sufficient evidence have been found for biofuel production and can met at-least 10% of energy demand for road transport of Dhaka city, Bangladesh. Without compromising with food security the study suggests that it is possible to significantly increase the biofuel production, and to do this as an integrated part of the existing society also contributing with positive societal synergies.