Antibiotic resistance represents a serious global health concern and has stimulated the development of antimicrobial nanomaterials to combat resistant bacteria. Protein-based nanoparticles combining characteristics of both proteins and nanoparticles offer advantages including high biocompatibility, attractive biodegradability, enhanced bioavailability and functional versatility. They have played an increasing role as promising candidates for broad applications ranging from biocatalysts and drug delivery to vaccine development to cancer therapeutics. However, their application as antibacterial biomaterials to address challenging antibiotic-resistance problems has not been explicitly pursued. Herein, we describe engineering protein-only nanoparticles against resistant Gram-positive bacteria. A self-assembling peptide (P4) enables the assembly of a phage lytic enzyme (P128) into nanoparticles in response to pH reduction. Compared to native P128 and monomeric P4-P128, P128 nanoparticles (P128) demonstrated a stronger bactericidal ability with high potency at lower concentrations (2-3-fold lower), particularly for methicillin-resistant strains. In addition, P128 showed an enhanced thermal (up to 65 °C) and storage stability and elicited extensive damages to bacterial cell walls. These remarkable antibacterial abilities are likely due to the P128 nanostructure, mediating multivalent interactions with bacterial cell walls at increased local concentrations of endolysin. The engineered endolysin nanoparticles offer a promising antimicrobial alternative to conventional antibiotics.