Due to the global infection problem caused by the abuse of antibiotics, the preparation of novel antibacterial nanomaterials is a key and basic requirement for applications in antibacterial and bacterial imaging fields. This paper reports the one-step preparation of blue-green-emitting carbon dots (CDs) under low temperature (80 °C) with glucose as the carbon source, citric acid as the dehydrating agent, and polyethyleneimine as the nitrogen source. Through inhibition zone tests and minimum inhibitory concentration (MIC) experiments, the inhibitory abilities of prepared CDs against various microorganisms, including gram-positive bacteria, gram-negative bacteria and fungi, were compared. It is worth mentioning that the MIC of CDs against Staphylococcus aureus reaches 4.7 μg ml-1, and the CDs exhibit excellent biocompatibility. Moreover, studies on visual-treatment therapy, in which infection treatment can be performed at the same time as bacterial imaging, with the prepared functional antibacterial CDs based on fluorescence confocal imaging would be beneficial to their promising future in medical and biological fields.