- Antimicrobial peptides are an essential part of the innate immune system of most living things and the understanding of the biophysical properties and the different mechanisms of action are crucial for the de-novo development of simple and effective analogs. More specifically, antimicrobial lipopeptides have been gaining increased attention because of the pressure for new antimicrobial agents against resistant pathogens. The addition of a lipophilic fatty acid has proven to be an effective method to increase the association of a peptide with the membrane, thus increasing the biological activity of certain peptide sequences. Previously, we reported that linear ultrashort cationic lipopeptides even as short as 4 amino acids have potent antimicrobial and antifungal properties. We described the minimum peptide length, and fatty acid length necessary for activity. Also, innate-immunity-like peptides were described that contained multiple histidine residues. Although these peptides consisted of 12 to 15 amino acids, these were less toxic to the host, and were lytic to numerous pathogens and cancer cells at slightly acidic environments. Here we report the design of an ultrashort histidine containing peptide whose antifungal activity could be significantly increased in a covalent trimeric form. Low micromolar activity was observed for Aspergillus fumigatus and Cryptococcus neoformans but not Candida albecans. Using transmission electron microscopy, we observed that this trimeric ultrashort histidine containing peptide formed distinct and differing nanostructures at pH 5 and 7, which could explain the activity differences. Since various organs or areas of the human body have a slightly acidic pH environment such as tumors, gastric lumen and lung-lining fluids in cystic fibrosis and asthma, understanding the importance of nanostructure-activity relationships of these pH dependent ultrashort peptides could lead to improvements in the delivery and administration of the peptides.