In the present study, biodiesel was produced from a novel non edible oil source, Silybum marianum L. seeds oil (SMSO). The acid value of the extracted oil was found to be 13.60 mg KOH/g. Therefore, a dual step process viz. acid–base catalyzed transesterification was developed to produce the biodiesel. Free fatty acids in SMSO were converted into methyl esters through the esterification of the oil by using hydrochloric acid (HCl) as an acid catalyst with 6:1 methanol to molar ratio at 60°C for 1 h, to reduce the acid value of the parent oil to below (2 mg KOH/g of oil), a value safe to produce the biodiesel through alkaline catalyzed transesterification. During alkaline-catalyzed transesterification, the parameters of this step such as alkaline catalyst type and concentration, methanol to oil molar ratio, reaction temperature and reaction duration were optimized. The results of esterification indicated that 1.0% HCl w/w of oil was optimal for the esterification, because it lowers the acid value to the minimum. For alkaline transesterification, a maximum conversion of (95.0%) was obtained by using potassium hydroxide as a catalyst with a concentration of 0.90% w/w of oil, 6:1 methanol to oil molar ratio, 60 °C reaction temperature and a reaction duration of 100 min. Biodiesel was also analyzed by using thin layer chromatography and Fourier transform infra red spectroscopy. The properties of the produced biodiesel are found to conform with the ASTM standard specifications.