Antimicrobial peptides (AMPs) have attracted considerable attention because of their multiple and complex mechanisms of action toward resistant bacteria. However, reports have increasingly highlighted how bacteria can escape AMP administration. Here, we have described the use multiple strategies including genetic and Joker algorithms to design synthetic AMPs derived from bacteria, plants and animals. This approach yielded different peptides classes including mastoparans, glycin-rich and clavanins that possess an unusually high proportion of cationic residues and use tyrosine residues as hydrophobic counterparts. At least dozens of peptides emerged as a prototype AMP, among fifteen guavanin analogues screened for their activity against an engineered luminescent strain. Similarly, clavanins and mastoparans derivated were also selected. Peptides were further characterized in terms of structure, activity and biotechnological potential for development new compounds useful for human and animal health. Most of those novel peptides were unstructured in water and underwent a coil-to helix transition in hydrophobic environments. This conformation was corroborated by NMR analysis in dodecylphosphocholine micelles, which revealed an α-helical structure. Peptides generated caused a bactericidal effect at low micromolar concentrations to several resistant bacteria, causing membrane disruption, without triggering depolarization but rather hyperpolarization. Finally, here the strategies for production in large scale was also discussed in order to prepare such peptides for the market. In summary the present work presents a computational approach to explore natural products for the design of short and potent peptide antibiotics that could be used against resistant bacteria.