In our venture towards the development of effective cytotoxic agents, a panel of triazole linked 3-benzylidene isatin hybrids were synthesized and characterized by IR, 1H NMR, 13C NMR and Mass spectral analysis. All the newly synthesized target compounds were assessed against DU145 (prostate), PC-3 (prostate), MDA-MB-231 (breast), BT549 (breast), A549 (lung) and HeLa (cervical) human cancer cell lines by employing MTT assay for their cytotoxic potential. Significantly, compound Z-8l was found to be most potent amongst all the tested compounds with an IC50 value of (3.7 ± 0.05 μM) on DU145 cells. The most active compound (Z-8l) was also tested on RWPE-1 (normal prostate) cells and was found to be safe compared to the DU145 cells. The influence of the cytotoxic compound Z-8l on the cell cycle distribution was assessed on the DU145 cell line, exhibiting a cell cycle arrest at the G2/M phase. Additionally, treatment with compound Z-8l caused collapse of mitochondrial membrane potential (DΨm) in DU145 cells. Moreover, acridine orange/ethidium bromide staining, DAPI nuclear staining, DCFDA staining and annexin V binding assay confirmed that compound Z-8l can induce cell apoptosis in DU145 cells. Western blotting was performed to examine the appearance of active forms of cytochrome c, Bax, Bcl2 and PARP (Poly ADP ribose polymerase), indicator proteins of apoptosis in DU145 cells; the study confirmed the triggering of mitochondrial mediated apoptotic pathway upon exposure of compound Z-8l.