Staphylococcus aureus poses a severe public health problem as one of the vital causative agents of healthcare- and community-acquired infections. There is a globally urgent need for new drugs with a novel mode of action (MoA) to combat S. aureus biofilms and persisters that tolerate antibiotic treatment. We demonstrate that a benzonaphthopyranone glycoside, chrysomycin A (ChryA), is a rapid bactericide that is highly active against S. aureus persisters, robustly eradicates biofilms in vitro, and shows a sustainable killing efficacy in vivo. ChryA was suggested to target multiple critical cellular processes. A wide range of genetic and biochemical approaches showed that ChryA directly binds to GlmU and DapD, involved in the biosynthetic pathways for the cell wall peptidoglycan and lysine precursors, respectively, and inhibits the acetyltransferase activities by competition with their mutual substrate acetyl-CoA. Our study provides an effective antimicrobial strategy combining multiple MoAs onto a single small molecule for treatments of S. aureus persistent infections.