Antimicrobial peptides (AMPs) have the potential to become valuable antimicrobial drugs in the coming years since they offer wide spectrum of action, rapid bactericidal activity and low probability for resistance development in comparison with traditional antibiotics. The search and improvement of methodologies for the discovery of new antimicrobial peptides to treat resistant bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa are needed for further development of antimicrobial products. In this work, the software Peptide ID 1.0® was used to find new antimicrobial peptide candidates encrypted in proteins, considering the physicochemical parameters characteristic of antimicrobial peptides such as net positive net charge, hydrophobicity, sequence length, among others. From the selected protein fragments, new AMPs were designed after conservative and semi-conservative modifications and amidation of the C-terminal region. In vitro studies of the antimicrobial activity of the newly designed peptides showed that two peptides, P3-B and P3-C, were active against P. aeruginosa at low minimum inhibitory concentrations (MIC). Peptide P3-C had also activity against S. aureus. Furthermore, bactericidal activity and information on the possible mechanisms of action are described according to the scanning electron microscopy studies