The development of sustainable biofuels can help to reduce the reliance on fossil fuels and mitigate the impact of climate change. This study analyzes bioethanol production from agro-forestry residual biomass, namely eucalyptus residues and corn stover. The study includes process simulation using Aspen Plus software, followed by economic analysis and life cycle assessment (LCA) with the help of SimaPro software and by applying the environmental footprint (EF) 3.0 method. The economic analysis on the biorefinery’s economic viability, equipment, and production costs reveals a positive decision for bioethanol production from eucalyptus residues due to logistical and transportation costs. The minimum ethanol selling price (MESP) obtained was 2.19 €/L and 2.45 €/L for eucalyptus residues and corn stover, respectively. From the LCA with a functional unit of 1 MJ of ethanol, bioethanol production from eucalyptus residues results in a single score impact of 37.86 µPt, whereas for corn stover, it is 33.47 µPt. In the climate change impact category, the eucalyptus residues scenario has an impact of 0.264 kg CO2 eq/MJ ethanol while corn stover leads to 0.254 kg CO2 eq/MJ ethanol. In-situ enzyme production, heat integration, and the use of renewable energy sources were also analyzed. Combining in situ enzyme production with renewable energy sources lowers CO2 equivalent emissions by 89% for both feedstocks, in comparison to the base-case scenario.