Cathelicidin LL-37 belongs to the class of human defense peptides and is overexpressed in many cancers. Segments of LL-37 derived through biochemical processes have a wide range of activities. In this study, novel analogs of the 13-amino acid cathelicidin 17-29 amide segment FKRIVQRIKDFLR-NHwere prepared and examined for their antimicrobial and hemolytic activities, as well as for their cytotoxicity on cancer bronchial epithelial cells. Selected substitutions were performed on residues Rand Kin the hydrophilic side, Vand Fin the hydrophobic side of the interphase, and Fthat interacts with cell membranes. Specific motifs IIKK and LLKKL with anticancer and antimicrobial activities isolated from animals were also inserted into the 17-29 fragment to investigate how they affect activity. Substitution of the amino-terminal positive charge by acetylation and replacement of lysine by the aliphatic leucine in the peptide analog Ac-FKRIVQRILDFLR-NHresulted in significant cytotoxicity against A549 cancer cells with an ICvalue 3.90 μg/mL, with no cytotoxicity to human erythrocytes. The peptide Ac-FKRIVQIIKKFLR-NH, which incorporates the IIKK motif and the peptides FKRIVQLLKKLLR-NHand Ac-FKRIVQLLKKLLR-NH, which incorporate the LLKKL motif, displayed potent antimicrobial activity against gram-negative bacteria (MIC 3-7.5 μg/mL) and substantial cytotoxicity against bronchial epithelial cancer cells, (IC12.9-9.8 μg/mL), with no cytotoxic activity for human erythrocytes. The helical conformation of the synthetic peptides was confirmed by circular dichroism. Our study shows that appropriate substitutions, mainly in positions of the interphase, as well as the insertion of the motifs IIKK and LLKKL in the cathelicidin 17-29 segment, may lead to the preparation of effective biological compounds.