The development of sustainable agriculture and the increasing antibiotic resistance of human pathogens call for novel antimicrobial compounds. Here, we describe the extraction and characterization of a class of cationic circular lipopeptides, for which we propose the name relacidines, from the soil bacterium Brevibacillus laterosporus MG64. Relacidines are composed of a fatty acid side chain (4-methylhexanoic acid) and 13 amino acid residues. A lactone ring is formed by the last five amino acid residues and three positively charged ornithines are located in the linear fragment. Relacidines selectively combat Gram-negative pathogens, including phytopathogens and human pathogens. Further investigation of the mode of action revealed that relacidine B binds to the lipopolysaccharides but does not form pores in the cell membrane. We also provide proof to show that relacidine B does not affect the biosynthesis of the cell wall and RNA. Instead, it affects the oxidative phosphorylation process of cells and diminishes the biosynthesis of ATP. Transcription of relacidines is induced by plant pathogens, which strengthens the potential of B. laterosporus MG64 to be used as a biocontrol agent. Thus, we identified a new group of potent antibiotic compounds for combating Gram-negative pathogens of plants or animals.