ConspectusResistance of pathogenic bacteria against currently marketed antibiotics is again increasing. To meet the societal need for effective cures, scientists are faced with the challenge of developing more potent but equally bacteria-specific drugs. Currently, most efforts are directed toward the modification of existing antibiotics, but ideally, compounds with a new mode of action are required. In this Account, we detail our findings in the area of novel metal-based antibiotics. Our strategy is based on the modification of simple antimicrobial peptides (AMPs) with organometallic agents, resulting in organometallic AMPs (OM-AMPs). Since bacteria have most likely never encountered these synthetically prepared unnatural organometallic agents, we anticipated that such agents could well become potentiating players in the antibiotics arena. Moreover, exploiting some of the particular properties of metal complexes should also help to elucidate the mode of action of small cationic AMPs, the molecular details of which have remained elusive despite intensive efforts.Using standard Fmoc/tBu-based solid-phase peptide synthesis approaches, we have prepared various organometallic-peptide conjugates with covalently linked group 8 and 9 metallocenes (ferrocene, ruthenocene, osmocene, and cobaltocenium). As a starting point we took the (RW)3 antibacterial hexapeptide lead structure. After modifying the peptide sequence (generations 1 and 2), changing the nature and position of the organometallic group (generation 3), and optimizing the amino acid chirality (generation 5), we identified several organometallic antibacterial peptides that are currently among the most active synthetic AMPs (synAMPs) that have ever been prepared. Through these rational and systematic optimizations, we were able to increase the antibacterial activity of a short non-organometallic synAMP 18-fold to submicromolar activity, rivaling the activity of vancomycin (often the drug of last resort) against methicillin-resistant Staphylococcus aureus (MRSA). Moreover, by making use of the unique physicochemical properties of ruthenocene, we were able to determine the mode of action of these short AMPs in unprecedented detail. We propose that the OM-AMP integrates into the bacterial membrane and changes its biophysical properties, which ultimately results in detachment of vital enzymes for respiration and cell-wall biosynthesis such as specifically cytochrome c and MurG from their locations in the membrane. Further explorations of these small OM-AMP derivatives that are summarized in this Account include lipid substitution, multivalent display of metalated di- or tripeptides on a trivalent scaffold with different linkers, and increasing the metal-to-peptide ratio such that every tryptophan in the (RW)3 scaffold is eventually replaced by a metalated lysine. While initial experiments with our OM-AMPs for systemic applications were largely disappointing, these OM-AMPs turned out to be potent antibiotics for topical applications. In this sense, two applications are described as examples in this Account, namely, bacterial decontamination of wastewater by reverse osmosis membranes (coated with our OM-AMPs by Cu-catalyzed azide-alkyne cycloaddition reaction) and synergistic activities of one of our synAMPs with colistin and tobramycin for the treatment of Pseudomonas aeruginosa infections that are associated with cystic fibrosis.