Increasing prevalence of bacteria that carries resistance towards conventional antibiotics has prompted the investigation into new compounds for bacterial intervention to ensure efficient infection control in the future. One group of potential lead structures for antibiotics is antimicrobial peptides due to their characteristics as naturally derived compounds with antimicrobial activity. In this study we aimed at characterizing the mechanism of action of a small set of in silico optimized peptides. Following determination of peptide activity against E. coli, S. aureus and P. aeruginosa, toxicity was assessed revealing meaningful selectivity indexes for the majority of the peptides. Investigation of the peptides effect on bacteria demonstrated a rapid growth inhibition with signs of bacterial lysis together with increased bacterial size. Both visual and quantitative assays clearly demonstrated bacterial membrane disruption after 10 minutes for the most active peptides. The membrane disrupting effect was verified by measuring the release of calcein from bacterial mimicking liposomes. This revealed the most active peptides as inducers of immediate release, indicating the kinetics of membrane permeabilization as an important determinant of bacterial activity. No well-defined secondary structure of the peptides could be determined using CD-spectroscopy in the presence of different liposomes mixtures, implying that there is no correlation between peptide secondary structure and the observed anti-bacterial and cytotoxic activity for this set of peptides. In conjunction, these findings provide strong indications of membrane disruption as the primary mechanism of bacterial growth inhibition for the tested peptides. This article is protected by copyright. All rights reserved.