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Hemocyanins present in the hemolymph of invertebrates are multifunctional proteins that are responsible for oxygen transport and play crucial roles in the immune system. They have also been identified as a source of antimicrobial peptides during infection in mollusks. Hemocyanin has also been identified in the cephalopod ancestor Nautilus, but antimicrobial peptides derived from the hemocyanin of <i>Nautilus pompilius</i> have not been reported. Here, the bactericidal activity of six predicted peptides from <i>N. pompilius</i> hemocyanin and seven mutant peptides was analyzed. Among those peptides, a mutant peptide with 15 amino acids (1RVFAGFLRHGIKRSR15), <i>Np</i>HM4, showed relatively high antibacterial activity. <i>Np</i>HM4 was determined to have typical antimicrobial peptide characteristics, including a positive charge (+5.25) and a high hydrophobic residue ratio (40%), and it was predicted to form an alpha-helical structure. In addition, <i>Np</i>HM4 exhibited significant antibacterial activity against Gram-negative bacteria (MBC = 30 μM for <i>Vibrio alginolyticus</i>), with no cytotoxicity to mammalian cells even at a high concentration of 180 µM. Upon contact with <i>V. alginolyticus</i> cells, we confirmed that the bactericidal activity of <i>Np</i>HM4 was coupled with membrane permeabilization, which was further confirmed via ultrastructural images using a scanning electron microscope. Therefore, our study provides a rationalization for the development and optimization of antimicrobial peptide from the cephalopod ancestor Nautilus, paving the way for future novel AMP development with broad applications.