Pedobacter cryoconitis strain UP508 was isolated from a soil sample using a mixture of ampicillin, kanamycin, and nalidixic acid for selection. UP508 was found to produce >30 unknown antibacterial peptides, of which eight, isopedopeptins A-H (1-8), were isolated by bioassay-guided fractionation and characterized with respect to structures and biological properties. Compounds 1-8 were all composed of nine amino acid residues and one 3-hydroxy fatty acid residue, and the structures were ring-closed via an ester bond from the C-terminal aspartic acid to the 3-hydroxy fatty acid. The differences between the peptides were the size and branching of the 3-hydroxy fatty acid and the presence of a valine or a 3-hydroxyvaline residue. The isopedopeptins mainly had activity against Gram-negative bacteria, and isopedopeptin B (2), which had the best combination of antibacterial activity, in vitro cytotoxicity, and hemolytic properties, was selected for further studies against a larger panel of Gram-negative bacteria. Isopedopeptin B was found to have good activity against strains of WHO top-priority Gram-negative bacteria, i.e., carbapenem-resistant Acinetobacter baumannii, Escherichia coli, and Pseudomonas aeruginosa, with minimal inhibitory concentrations (MIC) down to 1, 2, and 4 μg/mL, respectively. Furthermore, compound 2 had activity against colistin-resistant strains of A. baumannii, E. coli, and Klebsiella pneumoniae, with a MIC down to 8, 2, and 4 μg/mL, respectively. Compound 6 was tested in an E. coli liposome system where it induced significant leakage, indicating membrane disruption as one mechanism involved in isopedopeptin antibacterial activity. Isopedopeptin B stands out as a promising candidate for further studies with the goal to develop a new antibiotic drug.