Evaluation of the cytotoxicity of novel octahydropyrazin[2,1-a:5,4-a′]diisoquinoline derivatives (1a–2c) employing a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and inhibition of [³H]thymidine incorporation into DNA demonstrated that these compounds were more active than etoposide and camptothecin in both MDA-MB-231 and MCF-7 human breast cancer cells. Flow cytometric analysis after Annexin V-FITC and propidium iodide staining also confirmed that apoptosis was the main response of human breast cancer cells to 1a–2c treatment. Our results suggest that apoptosis of human breast cancer cells in the presence of 1a–2c follows the mitochondrial pathway, with the decrease in mitochondrial membrane potential and activation of caspase 9, as well as by the external pathway with the significant increase in caspase 8 expression. Cytotoxic properties of compounds 1a–2c in cultured human breast cancer cells correlate to their ability to inhibit topoisomerase I/II.