Carbon dots (CDs) have recently emerged as antibacterial agents and have attracted considerable attention owing to their fascinating merits of small size, facile fabrication, and surface functionalization. Most of them are involved in external light activation or hybridization with other functional nanomaterials. Herein, we present peroxidase‐like Cu‐doped CDs (Cu‐CDs) for in vitro antibacterial applications. The unique peroxidase‐mimicking property of the Cu‐CDs was demonstrated by tetramethylbenzidine chromogenic assay, electron paramagnetic resonance spectra, and hydroxy radical probe. Escherichia coli and Staphylococcus aureus were chosen as representative gram‐negative/positive models against which Cu‐CDs exhibited superior antimicrobial activity even at a dosage down to 5 μg/mL. A possible mechanism of action was that the Cu‐CDs triggered a catalytic redox reaction of endogenous H2O2 and glutathione depletion in the bacteria cells, with subsequent oxidative stress and membrane disruption. This work provides a new strategy for the design of microenvironment‐responsive antimicrobial nano‐agents.

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