| Summary: | <i>Callerya speciosa</i> is widely distributed in tropical and subtropical countries and is traditionally used for preventing numerous disorders. In this study, a bioguided fractionation of ethyl acetate extract (SE) from <i>C. speciosa</i> root was carried out to target antioxidant and cytotoxic activities. Of the four fractions (SE1-SE4) obtained by column chromatography, SE4 had the strongest anti-radical ability in the DPPH and ABTS assays (IC<sub>50</sub> = 0.05 and 0.17 mg/mL, respectively), with results close to butylated hydroxytoluene (BHT), a common antioxidant agent. The cytotoxic activities against the selected cells were analyzed in this study by MTT assay. Accordingly, SE2, SE3, and SE4 significantly inhibited the viability of multiple myeloma cell lines, comprising U266 (IC<sub>50</sub> = 0.38, 0.09, and 0.11 mg/mL, respectively) and KMS11 (IC<sub>50</sub> = 0.09, 0.17, and 0.15 mg/mL, respectively), mantle cell lymphoma Mino (IC<sub>50</sub> = 0.08, 0.16, and 0.15 mg/mL, respectively), and the noncancerous cell line LCL (IC<sub>50</sub> = 0.40, 0.32, and 0.21 mg/mL, respectively). At a concentration of 125 µg/mL, SE2, SE3, and SE4 induced the cell apoptosis of U266 (32.2%, 53.2%, and 55.6%, respectively), KMS11 (36.9%, 40.8%, and 47.9%, respectively), Mino (36.6%, 39.8%, and 22.0%, respectively), and LCL (12.4%, 17.5%, and 23.5%, respectively) via annexin V assay. The dominant compounds detected in fractions by high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry (HPLC-ESI-MS/MS), were identified as isoflavones. This is the first report describing <i>C. speciosa</i> as a promising natural source of antileukemia and antimyeloma agents, which may be useful for the development of blood cancer treatments.
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