Conjugated linoleic acid (CLA) and eicosapentaenoic acid (EPA) have been recognized for their physiological functions and potential as dietary supplements. This study is aimed at investigating the production and enhancing the efficiency of these two acids from Lactiplantibacillus plantarum using sesame waste as a natural, cost-effective, and readily available culture medium. Response surface methodology (RSM) was employed to optimize the production of CLA and EPA in a solid bed system using sesame waste paste as the fermentation substrate. The main processing parameters, including three humidity levels (60%, 70%, and 80%), three inoculum percentages (2%, 4%, and 6% v/v), and three fermentation temperatures (30°C, 37°C, and 44°C), were optimized. An experiment was conducted to validate the determined conditions, and a strong correlation was observed between the experimental results and the predictions made by the software. The optimal conditions for CLA and EPA production were determined to be an inoculation level of 6% with 80% humidity and at 37°C. Gas chromatography analysis of the fermented sesame waste medium revealed that the highest yields of CLAc9t11 and CLAt10c12, as well as eicosapentaenoic acid, were obtained in the medium fermented with 80% humidity and 6% inoculation at 37°C. The respective percentages of these fatty acids in the total fatty acid composition were found to be 0.351% (w/v) and 0.1% (w/v) for CLA and 0.139% (w/v) for EPA under the optimized conditions. These findings contribute to the understanding of CLA and EPA production and highlight the potential of L. plantarum and sesame waste as a fermentation substrate for their efficient production.