OBJECTIVE: This study aimed to investigate the possible therapeutic benefits of Benincasa hispida extract as an adjunctive treatment for breast cancer. While previous studies have highlighted the anticancer effects of various components of Benincasa hispida, including its fruit and leaves, there remains a paucity of research concerning the application of Benincasa hispida seeds in the context of cancer treatment, especially breast cancer. METHODS: The cytotoxic impact of Benincasa hispida on breast cancer cell lines was assessed via in-vitro analysis employing the MTT assay. The apoptosis levels were assessed by applying the Annexin V assay. Furthermore, an analysis of the cell cycle and quantification of intracellular reactive oxygen species (ROS) were conducted. RESULTS: The findings of this study demonstrated noteworthy anti-proliferative properties of the Benincasa hispida extract, highlighting its ability to induce apoptotic cell death and to cause cell cycle arrest at the S-phase. Furthermore, the extract exhibited antioxidant characteristics, influencing reactive oxygen species. The aforementioned attributes present significant advantages in the restoration of disrupted physiological mechanisms within breast cancer cells, thereby suggesting potential therapeutic benefits for the treatment of breast cancer. CONCLUSION: In summary, the findings indicate that Benincasa hispida extract presents a noteworthy potential as a supplementary strategy in the treatment of breast cancer. A more thorough examination of the active components and the intricate mechanisms of action is necessary. Such efforts may ultimately promote the advancement of secure and effective therapeutic strategies for the management of breast cancer.