This article is confined to biodiesel production and characterization of fatty acid methyl esters synthesized from a novel source of non edible neem oil. Fatty acid methyl esters were produced through methlanolysis of neem oil by using methanol in the presence of NaOH and KOH as catalysts. Conversion of crude neem oil to biodiesel was 76.8%. Fuel properties of neem oil biodiesel (100%), i.e., flash point (124 degrees C), viscosity (4.91 at 40 degrees C), density (0.8785 at 15 degrees C Kg/L), color (2.5) were close to ASTM biodiesel standards. Similarly, fuel properties of biodiesel blends were also in accordance with ASTM standards. Quality and quantity of neem oil biodiesel, based on physico-chemical characters were determined by using analytical techniques, such as chromatography and mass spectrometry, nuclear magnetic resonance, and Fourier transform infrared analysis. It is concluded that the rise in the use of non edible oil biodiesel is inevitable and neem is the potential source in this scenario for a promising source of renewable energy.