Increasing interest in bioenergy production in the context of the transition towards a circular economy and the promotion of renewable energy has produced demands for optimization of the value chain of energy production to improve the environmental viability of the system. Hotspot analysis based on life cycle assessment (LCA) contributes to the mitigation of environmental burdens and is a very important step towards the implementation of a bioeconomy strategy. In this study, hotspots identified using two parallel pathways: a literature review and empirical research on four different biogas plants located in Poland. LCA and energy return on investment (EROI) analysis of the whole bioenergy production chain were considered to identify unit processes or activities that are highly damaging to the environment. The biogas plants differ mainly in the type of raw materials used as an input and in the method of delivery. The results show that the most impactful processes are those in the delivery of biomass, especially road transport by tractor. The second contributor was crop cultivation, where fossil fuels are also used. Although the EROI analysis indicates a negligible impact of transport on the energy efficiency of bioenergy plants, the environmental burden of biomass transportation should be taken into consideration when planning further measures to support the development of the bioeconomy.