The essential oil extracted from Tenebrio molitor larvae (EOTM) is a natural product containing trace elements with potential therapeutic properties. This study aimed to assess the anticancer effects of EOTM and its synergistic interactions with zoledronic acid, a bisphosphonate drug, on prostate cancer cell lines. The chemical composition of EOTM was analyzed using GC-MS revealing a high concentration of fatty acids. The cytotoxicity of EOTM, both as a standalone treatment and in combination with zoledronic acid, was evaluated on prostate cancer cell lines (LNCaP, PC3) and normal hSKM using MTT assays. Results demonstrated that EOTM exhibited selective toxicity, inhibiting the growth of cancer cells in a dose-dependent manner while sparing normal cells. Morphological assessments and gene expression analyses of BCL2 and BAX were conducted through microscopy, Western blotting, and real-time RT-qPCR. These analyses indicated that EOTM induced apoptosis in cancer cells, as evidenced by cellular shrinkage, membrane blebbing, and nuclear fragmentation. Western blot results showed that EOTM downregulated the anti-apoptotic protein BCL2 and upregulated the proapoptotic protein BAX, suggesting activation of apoptosis pathways. Additionally, the combination of EOTM with zoledronic acid amplified these effects. Hoechst 33258 staining further confirmed the purity of cells following treatment. In conclusion, EOTM exhibits strong anticancer properties by inducing apoptosis in prostate cancer cells and demonstrates synergistic potential when combined with zoledronic acid. These findings warrant further investigation of EOTM as a natural and effective cancer treatment option.