The rising tide of bacterial drug resistance has sparked renewed interest in bacteriophages, the natural predators of bacteria. Our study highlights IME-EFm1, a Caudoviricetes bacteriophage specifically targeting Enterococcus faecium. Through our investigations, we identified that the gene IME-EFm1-ORF24 encodes an amidase, referred to as gp24, with promising lytic capabilities. Remarkably, gp24 exhibited a wider lytic spectrum than its parent phage, successfully lysing 26 out of 32 E. faecium strains, compared to the phage's ability to lyse only 21. This protein demonstrated robust antibacterial activity, remaining effective at temperatures between 25 °C and 60 °C and across a pH range of 6 to 12. Additionally, gp24 displayed significant anti-biofilm properties, effectively dismantling established biofilms in vitro. In a mouse model of abdominal infection, gp24 achieved a 75% protection rate against a dose of 2 × 109 colony-forming units of E. faecium En383, significantly outperforming the control group (p < 0.05). These compelling results suggest that gp24 holds great potential as a novel antimicrobial agent for treating E. faecium infections.