Enterococcus faecalis is a Gram-positive opportunistic pathogen that causes nosocomial infections in humans. Due to the growing threat of antibiotic resistance of E. faecalis, bacteriophage therapy is a promising option for treating of E. faecalis infection. Here, we characterized a lytic E. faecalis bacteriophage vB_EfaS_efap05-1 with a dsDNA genome of 56,563 bp. Phage vB_EfaS_efap05-1 had a prolate head and a tail, and belongs to Saphexavirus which is a member of Siphoviridae. Efap05-1 uses either surface polysaccharide or membrane protein ComEA as the receptor because the mutation of both genes (ComEA and UDP-glucose 4-epimerase galE) prevents phage adsorption and leads to phage resistance, and complementation of ComEA or galE could recover its phage sensitivity. Our results provided a comprehensive analysis of a new E. faecalis phage and suggest efap05-1 as a potential antimicrobial agent.