Carbon dots (CDs) have a profound effect on elimination of bacteria, fungi, and viruses, but the lack of an exact mechanism to interact with bacterial cells limits their development. Herein, we separated the CDs derived from chlorhexidine gluconate into three groups with uniformly small-scale, middle-scale, and large-scale particle sizes by using different molecular weight cut-off membranes. These positively charged particles exhibit significant antibacterial activity against the Gram-negative bacteria Escherichia coli and the Gram-positive bacteria Staphylococcus aureus; they can cause an increase in bacterial cell permeability, synergistic destabilization, and broken integrity of the plasma membrane. Impressively, we found that antibacterial activity increases as the size of the CDs decreases. This phenomenon may stem from the differences in cellular uptake and distribution of CDs in the plasma membrane or restriction between the polar functional group and DNA molecule. Our study of the size effect as a target may improve the understanding of killing microorganisms by antibacterial CD drugs. (C) 2020 Elsevier Inc. All rights reserved.