Paenibacillus elgii BC34-6 was isolated from the red clay soil and showed highantifungal activity against various plant pathogenic fungi. The culture supernatant of the bacteria contains various pelgipeptin lipopeptides which were identified by LC-MS analysis. Pelgipeptins consist of pelgipeptin A, B, C and D, and are composed of 9 amino acids with short fatty acid chains. Although pelgipeptins have been known as effective antimicrobial peptides, the molecular structures and exact functional mechanism are unclear. Therefore, to investigate the structure and antimicrobial mechanism, we purified four pelgipeptins (A, B, C and D) from the culture media by various extraction procedures and chromatography. The sequence analyses of the purified peptides were carried out using LC-MS/MS, and the secondary structure characteristics were confirmed using CD spectroscopy. The complete molecular structure of pelgipeptins were solved by 2D NMR spectroscopy including TOCSY, DQF-COSY, NOESY, and HSQC spectra. Antimicrobial activities and molecular mechanism were investigated using determination of minimal inhibitory concentration (MIC), lipid depolarization, dye leakage, and hemolysis assay. The MICs of the purified peptides against various bacteria were determined using a microbroth dilution method in 96-multiwell microtiter plates. Pelgipeptins were highly active against gram-positive, gram-negative, and methicillin resistant staphylococcus aureus (MRSA) strains with 2–8 ㎍ml-1 MICs. The dye leakage and membrane depolarization profile of pelgipeptins exhibited a gradual increase in the dye release. The cytotoxicity of pelgipeptins using red blood cell with various mechanism studies suggested that pelgipeptin A is the most potential antibiotics with low cytotoxicity and high antimicrobial activity among pelgipeptin family.