The rapid growth in industrialization steadily increased the energy demand. The world’s population ultimately depends on petroleum as a major share of fuel for transportation and industrialization. Even though it is widely used in various sectors, its emission into the atmosphere creates serious problems in the form of acid rain, smog, etc. This present experimental investigation highlights the utilization of Vachellia nilotica seed oil methyl ester (VNSOME) synthesized from Vachellia nilotica seed oil (VNSO) fueled in a diesel engine to assess the emission and performance characteristics. VNSOME is produced using the alkaline catalyst (NaOH) transesterification technique. Four different fuel blends of biodiesel, namely, VNSOME5, VNSOME10, VNSOME15, and VNSOME20, were prepared and fueled in an unmodified engine. The engine brake thermal efficiency is lower, the brake-specific fuel consumption (BSFC) using VNSOME20 is higher, and the temperature of exhaust gas emitted after combustion is increased. The thermal efficiency is reduced by 7.34% with increased BSFC and exhaust gas temperature (EGT) of 9.3 and 14.28%, respectively, as compared to diesel fuel. Similarly, using an optimized biodiesel blend (VNSOME20), the emission emitted such as HC and CO is reduced by 19.14 and 22.2%, respectively. However, the engine fueled with the VNSOME20 biodiesel blend increased the level of CO2 and NO x emitted into the atmosphere when compared to diesel fuel.