This study investigates the anticancer potential of isocordoin, a prenylated chalcone found in Adesmia balsamica. In vitro assays on colorectal (HT-29), breast (MCF-7) and prostate (PC-3) cancer cell lines, together with a non-cancerous colon cell line (CoN CCD841), revealed that isocordoin is cytotoxic, with PC-3 and MCF-7 cells showing the highest sensitivity. The selectivity index was higher for PC-3 (5.2) than for MCF-7 (3.7) and HT-29 (2.9). Isocordoin induced morphological changes suggestive of apoptosis in tumor cells. Mechanistic studies on HT-29 and MCF-7 lines indicated that isocordoin might possess antioxidant properties while promoting the loss of mitochondrial membrane potential and caspases activation. Molecular docking showed a favorable interaction of isocordoin with caspase-3, which could explain its apoptotic effects. In silico predictions suggest that isocordoin has drug-like properties, including good absorption and permeability to the blood-brain barrier. The presence of the prenyl chain in isocordoin appears crucial for cytotoxic activity, supported by its higher lipophilicity and better interaction with caspase-3 compared to non-prenylated 2 ',4 '-dihydroxychalcone. Overall, isocordoin demonstrates promising anticancer activity, warranting further investigation as a potential therapeutic agent.