Biodiesel is considered a suitable substitute and eco-friendly energy source. It is produced from renewable sources. Second-generation feedstocks are gaining attention due to their availability in many parts of the world. The non-edible biomass reduces food competition in the market. The Xanthium spinosum is one of the unexplored non-edible biomass, which contains a high content of linoleic acid in its seeds. In this research, the potential of seed oil for methyl ester production has been analysed. It possesses a higher oil content of 35.30 ± 0.10 wt%. The total lipid and linoleic acid content of the seeds were found to be 35 and 32.11 wt%. The fuel properties of a Xanthium spinosum proved its potentiality as a biodiesel feedstock. To test the potential, the oil was converted into the methyl ester via transesterification with a yield of 93%. The different fuel properties like density, cetane number (CN), flash point, pour point and cloud point were in accordance with internationally recognised biodiesel standards and showed its better quality compared to others accounted biodiesel. As an alternative renewable energy source for biodiesel production, the oil from Xanthium spinosum biomass has the potential to be used as a greener, cleaner and sustainable approach.