Pelgipeptins are cyclic lipopeptides composed of nine amino acids with a short hydrophobic fatty acid chain. To date, pelgipeptin A, B, C, and D have been identified. Their structures are distinguished by amino acid composition and type of fatty acid chains. Here, we report a new pelgipeptin peptide (Pelgipeptin L, PGP-L) produced by Paenibacillus elgii BC34-6. The primary sequence of the purified PGP-L was determined by MS/MS analysis, and the secondary structural property was investigated using CD spectroscopy. The complete molecular structure of PGP-L was determined by 2D NMR spectroscopy. The sequence of PGP-L consists of 1Dab-2Val-3Dab-4Phe-5Leu-6Dab-7Val-8Leu-9Ser and a saturated linear fatty acid chain (-CH3CH2CH2CH3). In addition, the antimicrobial activity of PGP-L was determined by minimal inhibitory concentration (MIC). The mechanism of action of PGP-L was investigated by fluorescence experiments. In summary, PGP-L was highly active against Gram-negative, Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) with 2-8 of MICs (µg/mL). The calcein dye leakage and membrane depolarization profiles of PGP-L exhibited a gradual increase in the fluorescence intensity, which suggests that PGP-L is targeting bacterial membrane by membrane disruption. PGP-L showed lower hemolysis activity than other pelgipeptin peptides. Taken together, we suggest that PGP-L can be a potential antibiotics with low cytotoxicity.