The issue of bacterial drug resistance has been affecting the world, bringing risks to different fields such as food safety, medical hygiene, and environment. Thus, it is very important to develop effective antibacterial agents to replace antibiotics. Carbon nanomaterials have many advantages as antibacterial agents, including no problem of drug resistance, low environmental pollution, high antibacterial activity, low toxicity, and low cost. N-CQDs were evaluated as photosensitizer for Antimicrobial Photodynamic Therapy (aPDT) against bacteria. N-CQDs were synthesized by hydrothermal method using 1,3,6-trinitropyrene (TNP) as nitrogen source and carbon source. The antimicrobial test of N-CQDs was conducted against Gram-positive bacteria, Bacillus subtilis NCIB 3610, and Gram-negative bacteria including Staphylococcus aureus Newman, S. aureus USA300, and Escherichia coli K12 with different concentrations (0–100 ppm). The results showed that N-CQDs had an inhibiting effect on Gram-positive bacteria in the dark, but could not kill bacteria. However, compared to no-light incubation, the white light incubation with 20 W LED could kill bacteria and had better ROS production under 20 ppm N-CQDs. The minimal inhibition concentration (MIC) of two S. aureus Newman and USA300 was 20 ppm N-CQDs under light conditions, while that of B. subtilis was 100 ppm N-CQDs. Therefore, the results showed that N-CQDs had higher antimicrobial activity, and may be used as photosensitizer in aPDT and as antibacterial agents in food packaging and biomedical applications.