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Biodiesel represents a promising solution for sustainable energy needs, offering an eco-friendly alternative to conventional fossil fuels. In this research, we investigate the use of a catalyst derived from mussel shells to facilitate biodiesel production from Jatropha curcas oil. Our findings from X-ray Fluorescence (XRF) analysis emphasize the importance of carefully selecting calcination temperatures for mussel shell-based catalysts, with 1100 °C identified as optimal for maximizing CaO content. We identify a reaction time of 6 h as potentially optimal, with a reaction temperature of approximately 110 °C yielding the desired methyl ester composition. Notably, a methanol-to-oil ratio of 18:1 is the most favorable condition, and the optimal methyl ester composition is achieved at a calcined catalyst temperature of 900 °C. We also assess the stability of the catalyst, demonstrating its potential for reuse up to five times. Additionally, a thorough analysis of J. curcas Methyl Ester (JCME) biodiesel properties confirmed compliance with industry standards, with variations attributed to the unique characteristics of JCME. Comparing homogeneous (NaOH) and heterogeneous (CaO) catalysts highlights the potential of environmentally sourced heterogeneous catalysts to replace their homogeneous counterparts while maintaining efficiency. Our study presents a novel approach to sustainable biodiesel production, outlining optimal conditions and catalyst stability and highlighting additional benefits compared with NaOH catalysts. Therefore, utilizing mussel shell waste for catalyst synthesis can efficiently eliminate waste and produce cost-effective catalysts.