Abstract: Background and Objective: Croton reflexifolius has been used in traditional medicine for treating cancer. Although one study reported cytotoxic activity, the active compounds have not been identified. Therefore, the purpose of this study was to isolate and identify at least one cytotoxic compound from C. reflexifolius through a bioassay-guided study using A549 cell line. Methodology: The cytotoxic effect was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results: The current study indicates that the leaves of this plant contain more than one compound with the capacity of decreasing the viability of cancerous cells. From the hexane extract was isolated polyalthic acid, resulting in an IC50 value of 34.90±0.72 μg mL–1. It also demonstrated cytotoxic activity on HCC827, MCF7, MDA-MB-231, HeLa and Colo 205 cancer cell lines (IC50 values = 15.9-45.9 Fg mL–1). Regarding A549 versus normal cells, polyalthic acid showed greater selectivity for growth inhibition but was less potent than cisplatin, the standard drug for chemotherapy. Conclusion: By demonstrating that polyalthic acid has the capacity to inhibit the viability of cancerous cells, these results support the traditional use of C. reflexifolius as an anticancer treatment.