Bioassay guided fractionation by brine shrimp lethality test led to isolation of a cytotoxic compound, goniothalamin, from the root and stem of Goniothalamus macrophyllus (Blume) Hook. f. & Thomson var. macrophyllus. Goniothalamin showed a promising cytotoxicity (SRB assay) against colon cancer cell line (IC50 = 0.51±0.02 µg/ml), breast cancer cell lines (IC50= 0.95±0.02 µg/ml) and lung carcinoma (IC50 = 3.51± 0.03 µg/ml). LDH assay of goniothalamin suggested that it had no toxicity on cell membrane. Cytotoxic evaluation of goniothalamin to normal cell revealed moderate toxicity against skin fibroblast (IC50 = 26.73± 1.92 µg/ml) and human fibroblast (IC50 = 11.99±0.15 µg/ml).