A series of new phenanthrene-9-benzimidazole conjugates has been synthesized by condensing phenanthrene aldehydes with various substituted o-phenylenediamines. The title compounds were evaluated for their in vitro cytotoxic potential against various human cancer cell lines like breast (BT-549), prostate (PC-3 and DU145), triple negative breast cancer (MDA-MB-453), and human colon cancer (HCT-116 and HCT-15) cells. Among the tested compounds, 10o displayed significant in vitro cytotoxic activity against PC-3 prostate cancer cells with an IC50 value of 6.32 ± 0.09 μM. Further, the cell cycle analysis indicated that it blocks G2/M phase of the cell cycle in a dose dependent manner. In order to determine the effect of the compound 10o on cell viability; phase contrast microscopy, AO/EB staining, DAPI staining, and DCFDA staining studies were performed. In these studies, apoptotic features were clearly observed indicating that the compound inhibited cell proliferation by apoptosis. JC-1 staining and annexin binding assays indicated the extent of apoptosis in PC-3 cells. Further, relative viscosity measurements and molecular docking studies indicated that these compounds bind to DNA by intercalation.