In recent years, Enterococcus faecalis has emerged as an important opportunistic nosocomial pathogen capable of causing dangerous infections. Therefore, there is an urgent need to develop novel antibacterial agents to control this pathogen. Bacteriophages have very effective bactericidal activity and several advantages over other antimicrobial agents and so far, no serious or irreversible side effects of phage therapy have been described. The objective of this study was to characterize a novel virulent bacteriophage phi 4D isolated from sewage. Electron microscopy revealed its resemblance to Myoviridae, with an isometric head (74 +/- A 4 nm) and a long contractile tail (164 +/- A 4 nm). The phi 4D phage genome was tested using pulsed-field gel electrophoresis and estimated to be 145 +/- A 2 kb. It exhibited short latent period (25 min) and a relatively small burst size (36 PFU/cell). Tests were conducted on the host range, multiplicities of infection (MOI), thermal stability, digestion of DNA by restriction enzymes, and proteomic analyses of this phage. The isolated phage was capable of infecting a wide spectrum of enterococcal strains. The results of these investigations indicate that phi 4D is similar to other Myoviridae bacteriophages (for example phi EF24C), which have been successfully used in phagotherapy.