Purpose <p>The increasing prevalence of multidrug-resistant infections, combined with a decline in the discovery of new antibiotics, poses a significant threat to public health. This study aimed to identify a potent antimicrobial peptide (AMP)-producing bacterial strain, investigate its efficacy under various physiological conditions, and reveal its mode of action.</p> Methods <p>Cell-free supernatants (CFS) from seven bacterial strains were screened for extracellular proteins with antimicrobial activity against <i>Acinetobacter baumannii</i> and <i>Pseudomonas aeruginosa</i>. The most effective proteins showing antimicrobial activity were evaluated for stability under various conditions, including pH, temperature, salt, and protease exposure. The mode of killing was investigated, and the AMPs were identified using mass spectrometry.</p> Results <p>Extracellular proteins from <i>Pseudomonas extremaustralis</i> KU02 and <i>Pseudomonas bubulae</i> KU04 exhibited potent antimicrobial activity against Gram-negative bacteria, with the proteins from <i>P</i>. <i>bubulae</i> KU04 showing outstanding performance across all parameters. The target bacterial cells lose their membrane integrity and biofilm-forming capabilities upon treatment. Membrane and cell wall compromization were corroborated by the detection of a substantial amount of nucleic acids released from bacterial cells. Electron microscopic analysis revealed the devastating impact of extracellular proteins on bacterial cell walls. Gel retardation assays demonstrated the potential to disrupt the genetic material. Proteomic analysis revealed 192 proteins, of which two AMPs with predominantly random coil structures were identified.</p> Conclusion <p>The extracellular proteins of <i>P</i>. <i>bubulae</i> KU04, containing two identified antimicrobial peptides, exhibited high stability and demonstrated a dual mechanism of action, causing damage to both the bacterial cell wall and genomic DNA.</p> Graphical Abstract <p></p>