The interaction of natural lipopeptide pseudofactin II with a series of doubly charged metal cations was examined by matrixassisted laserdesorption ionizationtime of flight (MALDI-TOF) mass spectrometry and molecular modelling. The molecular modelling for metal-pseudofactin II provides information on the metalpeptide binding sites. Overall, Mg2+, Ca2+ and Zn2+ favor the association with oxygen atoms spanning the peptide backbone, whereas Cu2+ is coordinated by three nitrogens. Circular dichroism (CD) results confirmed that Zn2+ and Cu2+ can disrupt the secondary structure of pseudofactin II at high concentrations, while Ca2+ and Mg2+ did not essentially affect the structure of the lipopeptide. Interestingly, our results showed that the addition of Zn2+ and Cu2+ helped smaller micelles to form larger micellar aggregates. Since pseudofactin II binds metals, we tested whether this phenomena was somehow related to its antimicrobial activity against Staphylococcus epidermidis and Proteus mirabilis. We found that the antimicrobial effect of pseudofactin II was increased by supplementation of culture media with all tested divalent metal ions. Finally, by using Gram-positive and Gram-negative bacteria we showed that the higher antimicrobial activity of metal complexes of pseudofactin II is attributed to the disruption of the cytoplasmic membrane.