Antibiotics are the molecules of choice to treat bacterial infections. However, because of the rapid emergence of drug-resistant bacteria, alternative modes of combating infections are being envisaged. Bacteriophages, which infect and lyse bacterial cells, may function as effective antimicrobial agents. Most bacteriophages produce their own peptidoglycan hydrolase called endolysin or lysin, which breaks down the cell wall of bacteria and aids in the release of newly assembled virions. In this article, we have discussed several findings that help us in understanding how endolysins are regulated. We observe that there is no common mechanism that is followed in all the cases. Many different modes of activity regulation have been observed in endolysins, including regulation of protein expression, translocation across the cell membrane, and post-translational modifications. These processes not only demonstrate how endolysins are made dependent on other accessory proteins and non-protein factors for their synthesis, translocation across the cytoplasmic membrane, and activity, but also show how autoregulation helps in maintaining the enzyme in an inactive form. Various regulatory mechanisms discussed in this paper are particularly applicable to endolysins. Nevertheless, a detailed study of these methods opens new avenues of investigation in the area of protein translocation system and the novel ways of enzyme activation and regulation in bacteria.