Multidrug-resistant (MDR) bacteria have become a growing threat to public health. The gram-positiveEnterococcus faeciumis classified by WHO as a high-priority pathogen among the global priority list of antibiotic-resistant bacteria. Peptidoglycan-degrading enzymes (PDEs), also known as enzybiotics, are useful bactericidal agents in the fight against resistant bacteria. In this work, a genome-based screening approach of the genome ofE. faeciumallowed the identification of a putative PDE gene with predictive amidase activity (EfAmi1; EC 3.5.1.28) in a prophage-integrated sequence.EfAmi1 is composed by two domains: a N-terminal Zn2+-dependentN-acetylmuramoyl-l-alanine amidase-2 (NALAA-2) domain and a C-terminal domain with unknown structure and function. The full-length gene ofEfAmi1 was cloned and expressed as a 6xHis-tagged protein inE. coli.EfAmi1 was produced as a soluble protein, purified, and its lytic and antimicrobial activities were investigated using turbidity reduction and Kirby–Bauer disk-diffusion assays against clinically isolated bacterial pathogens. The crystal structure of the N-terminal amidase-2 domain was determined using X-ray crystallography at 1.97 Å resolution. It adopts a globular fold with several α-helices surrounding a central five-stranded β-sheet. Sequence comparison revealed a cluster of conserved amino acids that defines a putative binding site for a buried zinc ion. The results of the present study suggest thatEfAmi1 displays high lytic and antimicrobial activity and may represent a promising new antimicrobial in the post-antibiotic era.