Poly(ADP-ribose) polymerase-1 (PARP-1) plays a crucial role in DNA damage repair and has been identified as a promising therapeutic target in cancer therapy. As a continuation of our efforts on the development of novel PARP-1 inhibitors with potent anticancer activity, a series of benzamide derivatives containing the benzamidophenyl and phenylacetamidophenyl scaffolds were designed and synthesized based on the structure optimization of our previously reported compound IX. All target compounds were screened for their in vitro antiproliferative activities against human colorectal cancer cells (HCT116, DLD-1 and SW480) and human normal colonic epithelial cells (NCM460). Among them, compound 13f exhibited the most potent anticancer activity against HCT116 cells and DLD-1 cells with IC50 = 0.30 μM and 2.83 μM, respectively. Moreover, 13f displayed significant selectivity in inhibiting HCT116 cancer cells over the normal NCM460 cells. Furthermore, 13f exhibited excellent PARP-1 inhibitory effect with IC50 = 0.25 nM. Besides, 13f was found to effectively inhibit colony formation and migration of HCT116 cells. Studies on the mechanisms revealed that 13f could arrest cell cycle at G2/M phase, accumulate DNA double-strand breaks, reduce mitochondrial membrane potential and ultimately induce apoptosis in HCT116 cells. In addition, molecular docking study indicated that 13f could combine firmly with the catalytic pocket of PARP-1 through multiple hydrogen bond interactions. Collectively, these findings demonstrated that 13f could serve as a promising anticancer candidate and deserves further investigation.