The present study aims to explore the potential applications of Blepharocalyx cruckshanksii bark and leaf extracts as a cytotoxic agent against in vitro cancer cell lines (MCF-7, PC-3 and HT-29) by using sulforhodamine B (SRB) assay. The cytotoxicity assay revealed that the ethyl acetate extract from the bark exhibited marked anticancer activity. The active extract was subjected to liquid-liquid partitioning by using hexane and ethyl acetate to obtain fractions based on their polarity. However, Fraction 4 (F4) was identified as the most effective of the series by displaying against all cancer cell lines a cytotoxicity close to antineoplastic agents assayed. Then, F4 was analyzed by gas chromatography–mass spectrometry (GC-MS) to identify their major components and to relate these components to the cytotoxic effect. The results obtained indicated that B. cruckshanksii bark have excellent cytotoxic activity and warrant further studies to isolate novel compounds for chemotherapeutic use.