In this Letter, we presented in vitro studies of antimicrobial activity of Gly-His-Lys conjugates that are important point in preliminary biological evaluation of their potential application in skin and tissue therapies. The novel compounds include the conjugation of fatty acids with a modification of the amino acid sequence in the primary structure of Gly-His-Lys (6i). All the compounds exhibited strong to moderate activity. Compound 1d had the most potent antimicrobial activity at MIC ranges 31.3-125.0μg/mL (against Escherichia coli spp. and Staphylococcus aureus spp.), 375.0-500.0μg/mL (against Pseudomonas aeruginosa spp.). Conjugate 5b expressed activity against Staphylococcus aureus spp. and Escherichia coli spp. at MIC ranges 250.0-500.0μg/mL and 62.5-125.0μg/mL, respectively. Both conjugates 1d and 5b possessed rapid bactericidal activity against Gram-positive bacteria at 2MIC or 4MIC. Conjugates 1b-c, 1e, 2a-b and 4b showed noticeable effect against both Gram-positive and Gram-negative bacteria. Compounds 1d, 1e and 2e were the most active against fungus.