Tryptophan and arginine-rich antimicrobial peptides (AMPs) are promising antimicrobials to combat the rapidly increasing threat of antimicrobial resistance. However, Trp-rich AMPs are hemolytic and undergo degradation upon exposure to light. Consequently, ultrashort His-based lipopeptides are gaining interest as replacements for the light-sensitive Trp-rich AMPs. In the peptide design, Arg is generally preferred over Lys due to its higher activity, despite Lys being more selective. In our structure-activity relationship study with Myr-His-His-NH₂, we found Orn, a Lys homolog, to be a more potent substitute for Arg. Orn has only three methylene units, which appears to be the optimum side chain length for efficient interaction with His and the potent activity of a His-based lipopeptide. Looking into the potential mechanism of one of the promising lipopeptides Myr-Orn-His-NH₂, it likely acts on bacteria by inhibiting cell wall formation. Simple modifications of Myr-Orn-His-NH₂ and temporin-SHf produced new ultrashort His-based lipopeptides with at least 30 times more potency than ampicillin against Escherichia coli and Staphylococcus aureus. Given this promising activity, future work should explore the pharmacokinetics and pharmacodynamics of these new lipopeptides.