Abstract: In the face of the increasingly severe challenge of antimicrobial resistance, the development of novel antimicrobial strategies is urgently needed. Tetrahedral DNA nanostructures （TDNs） have emerged as a research hotspot in nanoscale antimicrobial technology, owing to their distinctive biocompatibility, programmability, stability and cell membrane penetration ability. By modifying various functional molecules, such as antisense peptide nucleic acids, antimicrobial drugs, antimicrobial peptides and aptamers, TDNs can be assembled into efficient multifunctional antimicrobial systems. These nanosystems exert significant antimicrobial effects through synergistic action and can target and deliver drugs to the infection site, effectively inhibiting the growth of various bacteria and disrupting stubborn biofilms. In addition, detection platforms based on TDNs can also achieve rapid detection of bacteria. This paper reviews recent research progress on the antimicrobial applications of TDNs and discusses current challenges and future directions for development. By summarizing the state of the art in this field, this review aims to provide a reference for the development of novel DNA-based nano-antimicrobial agents.