Aims: In this work, we aimed to identify an effective treatment of infections caused by Enterococcus spp. strains resistant to conventional antibiotics. Methods and Results: We report the isolation and characterization of a new lytic bacteriophage, designated bacteriophage EFAP-1, that is capable of lysing Enterococcus faecalis bacteria that exhibit resistance to multiple antibiotics. EFAP-1 has low sequence similarity to all known bacteriophages. Transmission electron microscopy confirmed that EFAP-1 belongs to the Siphoviridae family. A putative lytic protein of EFAP-1, endolysin EFAL-1, is encoded in ORF 2 and was expressed in Escherichia coli. Recombinant EFAL-1 had broad-spectrum lytic activity against several Gram-positive pathogens, including Ent. faecalis and Enterococcus faecium. Conclusions: The complete genome sequence of the newly isolated enterococcal lytic phage was analysed, and it was demonstrated that its recombinant endolysin had broad lytic activity against various Gram-positive pathogens. Significance and Impact of the Study: Bacteriophage EFAP-1 and its lytic protein, EFAL-1, can be utilized as potent antimicrobial agents against Enterococcus spp. strains resistant to conventional antibiotics in hospital infections and also as environmental disinfectants to control disease-causing Enterococcus spp. in dairy farms.