Infections caused by Gram-negative bacteria still pose a clinical challenge. Although nanomaterials have been developed for antibacterial treatments, a systematic evaluation of the mechanisms and intervention models of antibacterial materials toward Gram-negative bacteria is still lacking. Herein, antibacterial quaternized carbon dots (QCDs) were synthesized via a one-step melting method using anhydrous citric acid and diallyl dimethyl ammonium chloride (DDA). The QCDs exhibited effective broad-spectrum antibacterial activity and enhanced inhibitory ability towards Gram-negative bacteria. The antibacterial mechanism of the QCDs with respect to Gram-negative bacteria was investigated through the characterization of bacterial morphology changes, the absorption modes of the QCDs on bacteria, and the potential generation of reactive oxygen species by the QCDs. The QCDs showed low toxicity in different cells, and did not cause hemolysis. The QCDs were administered via intraperitoneal injection to treat acute peritonitis in mice infected with E. coli. Routine blood examination, magnetic resonance imaging, and pathological analysis were undertaken and it was found that, similar to the positive control group treated with gentamicin sulfate, the QCDs exhibited a therapeutic effect that eliminated infection and inflammation. This study explores a controllable synthetic strategy for the synthesis of active carbon dots with antibacterial activity, a material that is a promising candidate for new treatments of Gram-negative bacterial infections.