Colorectal cancer (CRC) is one of the most common causes of cancer death in the world. Although genes are considered the most important causes that contribute to CRC, intestinal microorganisms are an important player. It is proved that microbial infection has a great role in colon cancer initiation and progression. This present study tries to evaluate the effectiveness of the nano defensin as an antibacterial agent and its mechanism of action against bacterial species related to colorectal cancer. Defensin was extracted and purified from fenugreek seeds, then the purified protein was immobilized on nano chitosan (nano defensin). The antibacterial activity of the prepared nano defensin was tested against the main six bacterial species related to the CRC, Helicobacter pylori, Escherichia coli, Bacteroides fragilis, Salmonella enterica, Fusobacterium nucleatum, and porphyromonas gingivails. The prepared nano defensin showed high antibacterial activity against the tested bacteria. The mechanism of action of nano defensin against the studied bacteria is represented in the destruction of the plasma membrane of the bacterial species and its genetic materials. Immobilization increased the defensin activity and stability protected it from hydrolysis enzymes that could be produced by the bacteria. Nano defensin is a promising safe efficient antibacterial agent against bacteria related to colorectal cancer.