Anticancer role of oxindole compounds is well documented. Here, we synthesized new derivatives of 3-hydroxy-2-oxindole functionalized at position 3 (1a-f) which are expected to have antiproliferative activity in cancer cells. Human prostate cancer cell line (DU145) was treated with the synthesized derivatives at 40-μM concentration for 24, 48, and 72 h. Compounds 1-ethyl-3-hydroxy-1,1',3,3'-tetrahydro-2H,2'H-3,3'-biindole-2,2'-dione (1d), 5-bromo-1-ethyl-3-hydroxy-1,1',3,3'-2H,2'H-3,3'-biindole-2,2'-dione (1e), and 5-chloro-1-ethyl-3-hydroxy-1,1',3,3'-tetrahydro-2H,2'H-3,3'-biindole-2,2'-dione (1f) were found to significantly reduce DU145 cell viability at 48 and 72 h whereas no significant changes were observed up to 24 h. The compounds 1e and 1f showed the most cytotoxicity effect and had a similar antiproliferative activity on DU145 cell line. They have halogen and ethyl substitutions at positions 5 and 1, respectively. The IC of compound 1e for DU145 and A375 cells at 48 h was determined. The apoptotic effects and cell cycle progression of compound 1e at 1/2 × IC (55 μM) concentration in DU145 cells were investigated by nuclei staining, comet assay, flow cytometry, and scanning electron microscopy (SEM). The results obtained showed that this compound increased the percentage of tail DNA, increased the occurrence of the sub-G1 phase, and induced G2M arrest and apoptosis in DU145 cells after exposure for 48 h to a 55-μM concentration. The SEM images revealed cell contraction at 24 h, cell condensation, plasma membrane blebbing, and formation of apoptotic bodies at 48 and 72 h. These observations suggest that the antiproliferative activity of compound 1e may be to induce apoptosis in DU145 cells.