The national biofuel policy of the Indian government support to put a great emphasis on the synthesis of biodiesel from the nonedible feedstock. A potential option in this regard is Neem oil which is found in abundance in India (~1,00,000 tonnes). The major hindrance to the commercialization of biodiesel is its shelf life due to poor oxidation and stability characteristics. The current work investigates the oxidation stability of Neem biodiesel as per EU standard (EN14214) using the Rancimat apparatus. The oxidation stability of 12.3 hours without doping any chemical antioxidant in the synthesized Neem biodiesel makes it highly suitable for commercial acceptance. The impact of pyrogallol (PY) antioxidant was also investigated on the shelf life of Neem biodiesel build on response surface methodology (RSM) in accordance with Box-Behnken Design (BBD). Moreover, the execution and excretion characteristic of the Neem biodiesel blends: B15, B25, B35, and B45 are investigated on direct injection automotive compression ignition engine. Based on this broad experimental investigation B15 blend can be used as a fuel to power the existing automotive compression ignition engine with no compromise in the execution and excretion characteristics of the engine. From the analysis of the data of life-cycle assessment of the Neem biodiesel, it was found that it possesses the global warming potential of 45.72gCO2-eq Functional Unit−1 which is six times lesser than mineral diesel. Moreover, the Neem plantation has a carbon dioxide isolation potential of around 10 t ha−1 year −1. All these factors make the Neem Biodiesel highly suitable for blending with mineral diesel to power the existing compression ignition engines. © 2021 Taylor & Francis Group, LLC.