This study presents a comparative Life Cycle Assessment (LCA) of urban buses powered by Diesel S10 with three fuel blends: B7 (7% biodiesel), B15 (15% biodiesel), and B100 (100% biodiesel). Employing a well-to-wheel approach, the analysis covers the extraction, production, distribution, and use of the fuels, as well as vehicle manufacturing and maintenance. The environmental impacts were quantified using the CML-IA and ReCiPe 2016 (Midpoint and Endpoint) methods. Results indicate that B100 significantly reduces Global Warming Potential, yet exhibits higher impacts in eutrophication, abiotic depletion, and ecotoxicity. Sensitivity analysis regarding vehicle occupancy revealed greater variability for B100. In conclusion, the optimal fuel choice depends on the prioritization of specific impact categories, providing insights for sustainable transportation policies.