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The seeds of Aframomum melegueta are used extensively in the Nigerian ethnomedicine for the management of cancer. This study therefore aimed at isolating and characterizing its cytotoxic constituents. Methanol extract of the seed was obtained through cold maceration, and it was further partitioned into n – hexane, dichloromethane and ethylacetate. The most active fraction was purified on repeated chromatographic techniques, using vacuum liquid chromatography (VLC), column (CC), and high-performance liquid chromatography (HPLC). The extract, purified fractions and isolated compounds were tested for their cytotoxic activities against the human Rhabdomyosarcoma (RD) and breast (MCF-7) cancer cell lines, using MTT assay. The crude extract and n-hexane fraction were found to be selectively cytotoxic to the cancer cell lines. Bioassay-guided fractionation of the n-hexane fraction led to the isolation of three compounds, which were identified as 6-shogaoal, 6-paradol, and 1-dehydro-6-gingerdione. 6-shogaoal demonstrated the highest cytotoxicity with CC50 values of 0.11 ± 0.02 and 0.25 ± 0.05ìg/mL against RD and MCF-7 cell lines respectively, and these were higher in activity when compared with cyclophosphamide (CC50 = 1.98 ± 0.15 and 0.71 ± 0.7ìg/mL). The presented data validates the ethnomedicinal use of A. melegueta in the treatment of cancer and is also indicative of the potential of 6-shogaol as an anticancer agent.