The antimicrobial peptide Ab-Cath, is a promising candidate for development as treatment for antimicrobial resistant (AMR) bacterial infections. Future clinical use is hampered by Ab-Cath's cationic peptidic nature and limited therapeutic window. Here, we evaluated hyaluronic acid-based nanogels for encapsulation of Ab-Cath to circumvent these limitations. Using microfluidics, monodispersed anionic nanogels of 156–232 nm encapsulating >99 % Ab-Cath were prepared. Unprecedented, lyophilization using polyvinyl alcohol and dextran-40 provided Ab-Cath nanogel protection and allowed easy dose adjustment. Lyophilized and redispersed Ab-Cath nanogels were as effective as Ab-Cath solution in killing AMR Staphylococcus aureus, Acinetobacter baumannii and Escherichia coli in biological fluids, and in reducing S. aureus and A. baumannii biofilms. Importantly, encapsulation of Ab-Cath in nanogels reduced Ab-Cath's cytotoxic effects on human fibroblasts by ≥10-fold. Moreover, cutaneous application of Ab-Cath nanogels eliminated bacteria colonizing 3D human skin. These findings affirm the use of nanogels to increase the selectivity index of antimicrobial peptides.