The innate immune system of most vertebrates includes a family of host defense peptides named cathelicidins. Avian cathelicidins are excellent candidates for antimicrobial drug development due to their broad spectrum of activity against various microorganisms and low toxicity. Because of their extraordinarily long lifespan, parrots are exposed to a variety of infectious diseases during their lifetime, making them an excellent subject for the study of their immune system. The aim of this study was to evaluate the antimicrobial, hemolytic and cytotoxic activity of parrot cathelicidins and to determine their mode of action. To conduct in vitro tests, we synthesized six peptides: two from Amazona guildingii, two from Eolophus roseicapilla, and two from Gallus gallus (as reference peptides). Our data demonstrated that even at the greatest concentration examined, the peptides had no effect on VERO cells and human erythrocytes. At lower concentrations, the peptides showed strong antimicrobial activity with MIC and MBC values ranging from 12.5 µM to 1.56 µM. However, none of the peptides showed activity against Candida albicans. We used Sytox Green and performed a 4-hours time-kill assay to investigate the mechanism of action. The kinetic assay showed that the peptides required between 20 and 60 min to kill Escherichia coli and that they operated by disturbing the bacterial membrane. In general, parrot cathelicidin-derived peptides showed potent antimicrobial activity and could be used as novel templates for antimicrobial drug development.