Abstract Anteriorly we found Haemocyanin-derived peptides in semi-purified fractions of mucus secretion of Achatina fulica that showed an inhibitory effect on S. aureus strains. Here, we applied in silico rational design strategy to obtain new potential Antimicrobial Peptides (AMPs) from A. fulica haemocyanin-derived peptides (AfH). Designed peptides were chemically synthetized using the Fmoc strategy and antimicrobial activity against E. coli and S. aureus strains was investigated using the broth microdilution method. Cytotoxic activity on Vero, HaCat, and human erythrocytes cells was also evaluated. The results showed that 15-residue synthetic peptides, alpha-helical and cationic, were those with the highest biological activity against Gram positive strains, with Minimum Inhibitory Concentrations (MIC) in the range of 20 to 30 µM. The positive selectivity index suggests a higher selectivity mainly on the microorganisms evaluated and not on eukaryotic cells. In this study, A. fulica hemocyanin turned out to be an appropriate protein model for the rational design of AMPs against bacteria of public health importance. Additional studies are required to evaluate the activity of the peptides on Gram negative bacteria.