Lipidation enhances the antimicrobial potential of cationic peptides by increasing amphiphilicity. The nuclear localization signal peptide PKKKRKV, although cell-penetrating, has weak intrinsic antimicrobial activity. This study evaluates whether conjugation with fatty acids of varying chain lengths (C16–C18) and saturation can improve its antimicrobial properties and elucidate the associated structure–activity relationships. Five PKKKRKV-based lipopeptides (LP-1–LP-5) were synthesized using Fmoc solid-phase peptide synthesis and conjugated with different fatty acids. The compounds were purified by RP-HPLC and characterized by mass spectrometry and NMR. Antimicrobial activity was assessed against Gram-positive bacteria (Staphylococcus epidermidis, Staphylococcus aureus), Gram-negative bacteria (Escherichia coli, Salmonella typhimurium), and the fungus Candida albicans using broth microdilution assays. In silico studies, including molecular docking, molecular dynamics simulations, and DFT calculations, were conducted to investigate potential mechanisms and electronic properties. Lipidation significantly improved antimicrobial activity. LP-5 (C18:1, unsaturated) showed the broadest spectrum, with MIC values of 11.59–23.19 µM against both Gram-positive and Gram-negative bacteria. LP-2 (C16:0, saturated) exhibited selective potency against E. coli (MIC = 6.96 µM), while LP-3 and LP-4 demonstrated moderate, broad-spectrum activity. Computational analyses indicated favorable target interactions, though they did not fully correlate with experimental results. LP-4 and LP-5 showed the smallest HOMO–LUMO gaps (∼0.111–0.112 eV), suggesting higher electronic reactivity. Overall, lipidation of PKKKRKV is a promising strategy for developing antimicrobial agents. Chain length and saturation significantly influence activity, with LP-5 emerging as the most promising broad-spectrum candidate. Further studies are required to validate mechanisms and assess toxicity. © 2026 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.