Haemocyanin-derived peptides were previously found in semi-purified fractions of mucus secretion from the snail Achatina fulica, which exhibited an inhibitory effect on Staphylococcus aureus strains. Here, an in silico rational design strategy was employed to generate new antimicrobial peptides (AMPs) from A. fulica haemocyanin-derived peptides (AfH). The designed peptides were chemically synthetized using the Fmoc strategy, and their antimicrobial activity against Escherichia coli and S. aureus strains was evaluated using the broth microdilution method. In addition, the cytotoxic activity on Vero, HaCat, and human erythrocyte cells was also determined. The results demonstrated that 15-residue alpha-helical and cationic synthetic peptides exhibited the highest biological activity against Gram-positive strains, with minimum inhibitory concentrations (MIC) in the range from 7.5 to 30 mM. The positive selectivity index suggests a higher selectivity, primarily on the microorganisms evaluated, but not on eukaryotic cells. In this study, A. fulica hemocyanin was identified as an appropriate protein model for the rational design of AMPs against bacteria of public health significance. Further studies are required to evaluate the activity of the peptides on Gram-negative bacteria other than E. coli. (c) 2024 Elsevier B.V. and Soci & eacute;t & eacute; Fran & ccedil;aise de Biochimie et Biologie Mol & eacute;culaire (SFBBM). All rights are reserved, including those for text and data mining, AI training, and similar technologies.