The emergence of multidrug-resistant bacteria creates an urgent need for alternative antibiotics with new mechanisms of action. In this study, we synthesized a novel type of antimicrobial agent, Acr₃-NLS, by conjugating hydrophobic acridines to the N-terminus of a nuclear localization sequence (NLS), a short cationic peptide. To further improve the antimicrobial activity of our agent, dimeric (Acr₃-NLS)₂ was simultaneously synthesized by joining two monomeric Acr₃-NLS together via a disulfide linker. Our results show that Acr₃-NLS and especially (Acr₃-NLS)₂ display significant antimicrobial activity against gram-negative and gram-positive bacteria compared to that of the NLS. Subsequently, the results derived from the study on the mechanism of action demonstrate that Acr₃-NLS and (Acr₃-NLS)₂ can kill bacteria by membrane disruption and DNA binding. The double targets–cell membrane and intracellular DNA–will reduce the risk of bacteria developing resistance to Acr₃-NLS and (Acr₃-NLS)₂. Overall, this study provides a novel strategy to design highly effective antimicrobial agents with a dual mode of action for infection treatment.