Carbon quantum dots derived from gentamicin sulfate are directly developed by facile calcination at different temperatures. A promising nanomaterial, CQD180, even shows a much superior antibacterial activity compared with the antibiotic counterpart and low drug resistance, by preserving the active structure of the starting materials and providing an additional antibacterial mode arising from the positive charge surface and induced reactive oxygen species simultaneously. Moreover, CQD180 can effectively disrupt mature Staphylococcus aureus biofilm and selectively eliminate bacteria over mammalian cells.