Peptides can serve as versatile therapeutics with a highly modular structure and tunable biophysical properties. In particular, the efficacy of peptide antibiotics against drug-resistant pathogens is of great promise, as few new classes of antibiotics are being developed to overcome the ever-increasing bacterial resistance to contemporary drugs. This work reports biophysical and antimicrobial studies of a designed library of ultrashort peptides that self-assemble into hydrogels at concentrations as low as 0.5% w/v in buffered saline, as confirmed by rheology. The hydrogels are constituted by β-sheet-rich nanofibril networks, as determined by biophysical techniques including spectroscopy (attenuated total reflectance Fourier transform infrared spectroscopy and Congo red binding assay), short- and wide-angle X-ray scattering, and electron microscopy. Both peptide solutions and self-assembled hydrogels show potent antimicrobial activity against S. aureus and Pseudomonas aeruginosa by membrane lysis. These peptides also displayed selectivity toward bacterial cells over human dermal fibroblasts in vitro, as determined from Live/Dead, scanning electron microscopy, and coculture assays. This work reports an antimicrobial self-assembling motif of only three residues comprising an aromatically acylated cationic d-Dab/Lys amino acid, a second cationic residue, and naphthylalanine that heavily influences the self-assembly of these peptides into hydrogels. The variations in the antimicrobial activity and self-assembly properties between analogues may have implications in future studies on the correlation between self-assembly and biological activity in ultrashort peptides.