In the present study, we sought to establish the effect of diverse structural-related hydroxycoumarins on the proliferation, cytotoxicity, and induction of apoptosis in promonocytic leukemic cells (U-937). The dihydroxylated coumarins, 7,8-dihydroxy-coumarin and esculetin, induced DNA fragmentation as well as characteristic morphological changes of programmed cell death in U-937 cells. With the aim to perform a structure-activity relationship study, the correlation between the physicochemical properties of the molecules and their pro-apoptotic activity was carried out. Results showed that the presence of two adjacent phenolic hydroxyl groups was the most important factor in terms of the SAR. The exposure of leukemic cells to 7,8-dihydroxy-coumarin evoked a phenoxyl radical generation that was detected by electron spin resonance spectroscopy. The present study suggests that reactive oxygen species generation plays a critical role in dihydroxycoumarin-induced apoptosis in U-937 cells. These findings further suggest that these compounds may have a potential therapeutic role in the treatment of hematological malignancies.