Antimicrobial peptides from edible insects may serve as a potentially significant group of food preservatives. In the present work, the mode of action of a novel antimicrobial peptide MDpep9 from Chinese traditional edible housefly larvae was investigated. MDpep9 was shown to bind to bacterial DNA from the results of gel retardation and fluorescence quenching experiments. Further investigations confirmed that MDpep9 could bind with the phosphate group of DNA and intercalate into the base pairs in a helix of DNA or locate in hydrophobic environment of DNA. The previous and present results demonstrated that MDpep9 has dual mechanisms of bactericidal activity: disrupting bacterial cell membranes and binding to bacterial genomic DNA to inhibit cellular functions, ultimately leading to cell death. The results of DNA-binding mode may be contributive in designing new and promising antimicrobial peptides for food preservatives.