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Antimicrobial resistance poses an escalating threat to human health, necessitating the development of novel antimicrobial agents capable of addressing challenges posed by antibiotic-resistant bacteria. Thanatin, a 21-amino acid β-hairpin insect antimicrobial peptide featuring a single disulfide bond, exhibits broad-spectrum antibacterial activity, particularly effective against multidrug-resistant strains. The outer membrane biosynthesis system is recognized as a critical vulnerability in antibiotic-resistant bacteria, which thanatin targets to exert its antimicrobial effects. This peptide holds significant promise for diverse applications. This review begins with an examination of the structure–activity relationship and synthesis methods of thanatin. Subsequently, it explores thanatin’s antimicrobial activity, detailing its various mechanisms of action. Finally, it discusses prospective clinical, environmental, food, and agricultural applications of thanatin, offering valuable insights for future research endeavors.