The increasing resistance of microbe to available antibiotic is a major concern, leading to seek for a promising new generation of antimicrobial compounds. Alternatives to classical antibiotics are needed and peptide antibiotics are promising as one of the choices. Two promising families of drugs that meet these criteria are host-defense cationic antimicrobial peptides (AMPs) and lipopeptides. Most lipopeptides consist of a short linear or cyclic peptide sequence, with either a net positive or negative charge and a long fatty acid (8-18 carbon atoms) attached at the N-terminus with covalent bond. Many studies have shown that an amphipathic structure and hydrophobicity of the peptide are important for the biological function of antimicrobial peptides. However, the factors that dictate their cell selectivity are not yet clear. In the present study, we investigate the activity of KYR sequence and the attachment of fatty acid with different lengths as decanoic acid (C10), lauric acid (C12), myristic acid (C14) and palmitic acid (C16) at the N-terminus of the KYR sequence to compare effect of aliphatic acid length and addition of hydrophobicity of ultrashort peptide activity. The results reveal that: (i) increase in chain length of Cx-KYR increased lytic activity against different type of microbes; (ii) In the time killing kinetic assay, C16-KYR at 20 µM could completely inhibit the growth of Staphylococcus aureus 1466 and Escherichia coli O157:H7 within 1 hour; (iii) All lipopeptides could depolarize the membrane of bacteria.