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Many essential enzymes in bacteria remain promising potential targets of antibacterial agents. In this study, we discovered that dequalinium, a topical antibacterial agent, is an inhibitor of Staphylococcus aureus primase DnaG (SaDnaG) with low-micromolar minimum inhibitory concentrations against several S. aureus strains, including methicillin-resistant bacteria. Mechanistic studies of dequalinium and a series of nine of its synthesized analogues revealed that these compounds are single-stranded DNA bisintercalators that penetrate a bac