In the last few decades, the prevalence of multidrug resistance and importunate virulence in bacteria emerged as one of the serious concerns in health-care system. Lantibiotics, the well-conserved antimicrobial peptides predominantly rich in atypical sulfur-containing amino acids, lanthionine (Lan), and 3-methyl-lanthionine (MeLan), synthesized ribosomally from bacterial cells and modified posttranslationally, have drawn considerable interest in the therapeutic management of infectious diseases because of their effectiveness against multidrug-resistant bacteria. So far studied, the other reasons remain their potency, broad-spectrum nature, and negligible cytotoxicity. The biosynthesis of the lantibiotics involves two major steps, the modification of precursor peptides and their proteolytic activation. The gene cluster constituting the biosynthetic machinery codes essentially for the prepeptide and modification enzymes besides the proteases, ATP-binding cassette transporters, immunity factors, and regulatory proteins required as accessory factors. Herein, in this chapter a comprehensive discussion has been made on the biosynthesis of lantibiotics, which may be noteworthy in biomedical science for technology advancement.