Virion-associated peptidoglycan hydrolases (VAPGH) are phage-encoded lytic enzymes that locally degrade the peptidoglycan (PG) of the bacterial cell wall during infection. In contrast to endolysins, PGHs that mediate lysis of the host bacteria at the end of the lytic cycle to release of phage progeny, the action of VAPGHs generates a small hole through which the phage tail tube crosses the cell envelope to eject the phage genetic material at the beginning to the infection cycle. The antimicrobial activity of VAPGHs was first discovered through the observation of the phenomenon of 'lysis from without', in which the disruption of the bacterial cell wall occurs prior to phage production and is caused by a high number of phages adsorbed onto the cell surface. Based on a unique combination of properties of VAPGHs such as high specificity, remarkable thermostability, and a modular organization, these proteins are potential candidates as new antibacterial agents, e.g. against antibiotic-resistant bacteria in human therapy and veterinary as well as biopreservatives in food safety, and as biocontrol agents of harmful bacteria in agriculture. This review provides an overview of the different VAPGHs discovered to date and their potential as novel antimicrobials.