Several antimicrobial peptides (AMPs) have been reported in amphibian toxins, as temporin‐PTa from Hylarana picturata. The amino acid distribution within a helical structure of AMPs favors the design of new bioactive peptides. Therefore, this work reports the rational design of two new synthetic peptides denominated Hp‐MAP1 and Hp‐MAP2 derived from temporin‐PTa. These peptides present an amphipathic helix with positive charges of +4 and +5, hydrophobic moment (<µH>) of 0.66 and 0.72 and hydrophobicity () of 0.49 and 0.41, respectively. Hp‐MAP1 and Hp‐MAP2 displayed in vitro activity against Gram‐negative and Gram‐positive bacteria from 2.8 to 92 µM, without presenting hemolytic effects. Molecular dynamics simulation suggested that the parent and designed temporin‐like peptides lack structural stability in an aqueous solution. By contrast, α‐helical structures were predicted in hydrophobic and anionic environments. Additionally, the peptides were simulated on mimetic membranes composed of anionic and neutral phospholipids 1,2‐dipalmitoylsn‐glycerol‐3‐phosphatidylglycerol (DPPG‐anionic), 1,2‐dipalmitoyl‐sn‐lyco‐3 phosphatidylethanolamine (DPPE‐neutral). When in contact with DPPG/DPPE (90:10) and DPPG/DPPE (50:50) temporin‐PTa, Hp‐MAP1 and Hp‐MAP2 established interactions guided by hydrogen and saline bounds. Therefore, the findings described here reveal that the optimization of the amphipathic α‐helical cationic peptides Hp‐MAP1 and Hp‐MAP2 enabled the generation of new synthetic antimicrobial agents to combat pathogenic microorganisms.