BACKGROUND: Colorectal cancer (CRC) is one of the most common malignancies worldwide, and despite advances in treatment, there remains a critical need for novel therapeutic approaches. Recently, anti-microbial peptides (AMPs) have gained attention for their potential use in cancer therapy due to their selective cytotoxicity towards cancer cells. OBJECTIVE: This study aims to evaluate the anti-cancer potential of two computationally engineered anti-microbial peptides (EAMPs) in SW620, SW480, and HCT116 colon cancer cells and the normal colon epithelial cell line CCD 841, focusing on their effects on cell proliferation, apoptosis, and DNA damage. METHOD: Cell proliferation and survival were measured using the CellTiter-Glo Luminescence and clonogenic assays. DNA damage was assessed through the Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Flow cytometry was used to examine cell apoptosis, cell cycle distribution, and mitochondrial membrane potential in SW620 cells. RESULTS: EAMPs inhibited CRC cell proliferation in a dose-dependent manner, with minimal toxicity observed in normal colon epithelial cells. In SW620 cells, EAMPs induced DNA damage, resulting in cell cycle arrest at the S/G2 phase, apoptosis, and a reduction in mitochondrial membrane potential. The proliferation results were confirmed in SW480 and HCT116 CRC cell lines. CONCLUSION: Our findings revealed that EAMPs exhibited significant anti-cancer activity against CRC cells in vitro while sparing normal epithelial cells. These results suggest that EAMPs may offer a potential therapeutic approach for colorectal cancer and warrant further investigation.