Novel antibacterial carbon dots (CDs), because of their excellent biocompatibility, biological activity, and advantages in imaging, have been a hot spot in recent studies. This paper reports the simple and fast preparation of multifunctional blue-emitting protamine sulfate (PS)-derived CDs (PS-CDs) through a one-step microwave-assisted method. The prepared PS-CDs exhibit advantages such as excellent water solubility, low cell toxicity and excellent blood compatibility. The minimum inhibitory concentration (MIC) test was used to systematically study the antimicrobial efficacy of the prepared PS-CDs against various microorganisms, including Gram-positive and Gram-negative bacteria and fungi. The produced PS-CDs exhibit strong antibacterial activity against Staphylococcus aureus (S. aureus) with a MIC of 25 μg mL⁻¹ and methicillin-resistant S. aureus (MRSA) with a MIC of 37.5 μg mL⁻¹. Additionally, PS-CDs show little difference in their sensitivity to S. aureus and drug-resistant bacteria but high sensitivity to the structural differences of Gram-negative bacteria. In addition, the antibacterial mechanism of these PS-CDs is proposed, and the application of these PS-CDs for imaging was realized, thanks to their easy internalization into bacteria. Compared to traditional antibacterial agents, both the bactericidal activity and biological imaging of PS-CDs demonstrate their high potential for applications in the biomedical field.