In this study, we assessed the cytotoxic effect of synthetic 2′,4′,5′-trimethoxychalcones on the human K562 acute myeloid leukemia cell and human Jurkat acute lymphoid leukemia cell. Compounds 13, 16, 19, and 26 showed low IC50 values (4.10–8.56 μM at 72 h) for both cell lines and did not have a cytotoxic effect on normal human lymphocytes. The mechanism of cell death induced by these compounds involves a decrease in the expression of cell proliferation marker Ki67, suggesting inhibition of cell proliferation. Furthermore, these chalcones reduced mitochondrial potential, decreased Bcl-2 expression, and increased Bax expression, indicating that the mechanism of apoptosis induced by them involves the intrinsic apoptosis pathway. The mechanism of action also involves increase in active caspase-3 and decrease in survivin expression. These results support the chalcones as potential antitumoral agents for further optimization.