Cage-shaped nucleic acid nanocarriers are promising molecular scaffolds for the organization of polypeptides. However, there is an unmet need for facile loading strategies that truly emulate nature's host-guest systems to drive encapsulation of antimicrobial peptides (AMPs) without loss of biological activity. Herein, we develop DNA nanogels with rapid in situ loading of L12 peptide during the thermal annealing process. By leveraging the binding affinity of L12 to the polyanionic core, we successfully confine the AMPs within the DNA nanogel. We report that the thermostability of L12 in parallel with the high encapsulation efficiency, low toxicity and sustained drug release of the pre-loaded L12 nanogels can be translated into significant antimicrobial activity. Using an S. aureus model of infectious bacterial keratitis, we observe fast resolution of clinical symptoms and significant reduction of bacterial bioburden. Collectively, this study paves the way for the development of DNA nanocarriers for caging AMPs with immense significance to address the rise of resistance.