A series of DHAA thiourea derivatives containing bisphosphonate moiety were designed and synthesized as potent antitumor agents. Structures of target molecules were confirmed using HR-MS, (1)H NMR and (13)C NMR and they exhibited potent anti-tumor activities against the SK-OV-3, BEL-7404, A549, HCT-116 and NCI-H460 tumor cell lines in vitro. Especially, compound 6e (IC50 = 1.79 ± 0.43 μM) exhibited the best anticancer activity against SK-OV-3 cell line. Its role as an inducer of apoptosis was investigated in this cell line by Annexin-V/PI binding assay and by following its capability for ROS generation, depolarization of mitochondrial transmembrane potential, activation of caspases and expression of pro- and anti-apoptotic proteins. Elevated level of ROS generation, activation of caspase-3, caspase-8, caspase-9, and Fas, higher expression of Bax, lower expression of Bcl-2, and increased level of Bax/Bcl-2 ratio identified 6e as a promising inducer of apoptosis that follows both of the mitochondria dependent pathway and the death receptor-mediated pathway. In addition, the cell cycle analysis indicated that compound 6e caused cell cycle arrest at G1 phase, induced apoptosis and led to cell death by increasing the proportion of sub-G1 cells. Furthermore, molecular docking studies showed that 6e could bind to the ATP pocket sites.