The exploration of natural sources for novel anticancer agents has garnered significant attention in recent years, driven by the need for effective and safe anticancer medications, considering the escalating global burden of cervical cancer. Vitex doniana Sweet (Verbenaceae), a plant with diverse traditional medicinal uses, especially in Africa, has shown promise in this regard due to its rich phytochemical composition. This study investigated the cytotoxic and antiproliferative effects of V. doniana extracts on normal mammalian (Vero-CCL-81) and cervical cancer (HeLa) cells, including cancer-associated gene expression profiles, according to standard procedures. Phytochemical analysis was performed using gas chromatography-mass spectrometry (GC-MS). The findings revealed significant (P < 0.0001) concentration-dependent increases in cytotoxicity and inhibition of cancer cell proliferation, by the aqueous, methanolic, ethyl acetate, and dichloromethane leaf extracts of V. doniana. Notably, differential cytotoxicity profiles were observed among different extracts. The median cytotoxic concentrations (CC These findings provide valuable insights into the potential and molecular mechanisms underlying the anti-cervical cancer effects of V. doniana extracts, their attributable phytocompounds, and highlight their putative value as sources of lead compounds for the development of novel anticancer drugs.