Contemporary medicine has been confronted by multidrug resistance. Therefore, new antibiotics are sought to alleviate the problem. In this study, we estimated the effect of the positioning and extent of lipidation (mainly octanoic acid residue) in the KR12-NHmolecule on antibacterial and hemolytic activities. The effect of the conjugation of benzoic acid derivatives (CH-X-COOH, where X: CH, CH-CH, CH=CH, C≡C, and CH-CH-CH) with the-terminal part of KR12-NHon biological activity was also studied. All analogs were tested against planktonic cells of ESKAPE bacteria and reference strains of. The effect of lipidation site on the helicity of the KR12-NHanalogs was studied using CD spectroscopy. The ability of the selected peptides to induce the aggregation of POPG liposomes was evaluated with DLS measurements. We demonstrated that both the site and extent of peptide lipidation play an essential role in the bacterial specificity of the lipopeptides. Most of the C-KR12-NH() analogs that were more hydrophobic than the parent compound were also more hemolytic. A similar relationship was also found between the α-helical structure content in POPC and hemolytic activity. It is worth emphasizing that in our study, the highest selectivity againststrains with an SI value of at least 21.11 exhibited peptideobtained by the conjugation of the octanoic acid with the-terminus of retro-KR12-NH. All lipidated analogs with the highest net charge (+5) were the most selective toward pathogens. Therefore, the overall charge of KR12-NHanalogs plays pivotal role in their biological activity.