Cathelicidin derived from human leukocytes is a 171amino acid protein that demonstrated to have antimicrobial activity. Synthesis of Antimicrobial Peptides (AMPs) are known to be expensive. In this study, after extraction of human cathelicidin sequence from NCBI, physicochemical characteristics such as length, amino acids composition, hydrophobicity and net charge were determined. Several truncated peptides were selected and probability of antimicrobial activity was predicted by Antimicrobial Peptide Prediction software. Purity and molecular weight of the peptides were confirmed by HPLC and mass spectrometry. Eventually, Minimum Inhibitory Concentration (MIC) of these peptides against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa were examined by micro-dilution method. Two peptides were synthesized as a result of bioinformatics method. One of the two prepared peptides with 13 amino acids length showed good activity against the bacteria. MIC results of this peptide were 15.6 μg/ml (against E. coli), 31.2 μg/ml (against P. aeruginosa) and 31.2 μg/ml (against S. aureus). Another peptide, Cth14, showed 125 μg/ml against E. coli. IC50 of Cth13 and Cth14 were 959 μg/ml and 335 μg/ml, respectively.