Renewable fuels have been used for years as an option for supplying energy demand in a sustainable manner. In terms of liquid fuels, hydrotreated vegetable oil (HVO) gained special attention in the last few years due to its good performance in diesel engines, low emissions and renewable aspect. At the same time, bioethanol represents another renewable option for energy supply and has been consolidated in the market for decades. Even though these biofuels are assumed to be environmentally friendly, a complete understanding of their environmental impacts should take into account several indicators, applications and geographical aspects. In this sense, this study presents a detailed Life Cycle Assessment (LCA) regarding the use of petrodiesel, HVO from palm and soybean oil and bioethanol in various operating conditions in a diesel engine, for small-scale electricity generation in stationary applications. Diesel and HVO are used as pilot fuels, while bioethanol was injected in the intake port in various Energy Fraction Ratios (EF), EF = 0%; EF = 28%; EF = 32%; EF = 40%; EF = 44%, in the so-called dual-fuel mode. The results obtained with pure HVO showed reductions in specific levels of NOx, HC, CO and particulate matter (PM) emissions of 30%, 75%, 81% and 55.3%, respectively, and 4.36% efficiency when compared to diesel. The Dual-Fuel application provided a significant reduction in CO2, NOX and PM emissions, but decreased engine efficiency by up to 9.4%. The LCA showed that the palm oil HVO has an environmental performance superior to the soybean HVO and is capable of reducing the Global Warming Potential up to 75%. Palm oil HVO reduces impacts in categories such as Terrestrial Acidification, Ozone Formation and Consumption of non-renewable resources when compared to diesel. The environmental impact of the dual-fuel operation presents a decrease in the levels of Global Warming Potential, Depletion of Fossil Resources and Ozone Formation.