In this research, negative-charge carbon dots (CDs) were synthesized in one-step using a microwave and found to have potential antibacterial ability against multi-drug resistant bacteria. The CDs were synthesized by using citric acid and urea as precursors, and characterized by FT-IR, TEM and fluorescence spectrophotometry. The average size of CDs was about 2.5 nm, and the ζ potential was −11.06 mV. In the following antibacterial activity test, time-killing curve experiments and colony-forming assay were carried out to determine the minimum bactericidal concentration (MBC) and minimum inhibitory concentration (MIC) of the CDs against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-intermediate Staphylococcus aureus (VISA). The data showed the MBC of the CDs against MRSA is 2.5 mg mL⁻¹, and the MIC of the CDs against MRSA is 0.63 mg mL⁻¹; the MBC of the CDs against VISA is 1.25 mg mL⁻¹, and the MIC of the CDs against VISA is 0.63 mg mL⁻¹. The results demonstrated that the negative-charge CDs have potential against multi-drug resistant Staphylococcus aureus (S. aureus), and may serve as alternatives for therapy in the future.