Vitamin C-based carbon dots are a new class of nanomaterials with antibacterial properties, but their antibacterial activities against aquatic pathogenic bacteria have not been verified. For this reason, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of V_C-CDs against 12 aquatic pathogenic bacteria such as Aeromonas hydrophila, Vibrio parahaemolyticus, V. splendens and Nocardia sp. were determined using 3-fold serial dilutions. The bacteriostatic kinetic curves of the 12 aquatic pathogens were determined. In addition, based on the potential biocompatibility of nano materials, we used MTT method to detect the cytotoxicity of V_C-CDs on CIK cells. Danio rerio embryos were used as test objects to determine the embryotoxicity after exposure to V_C-CDs. The results showed that V_C-CDs had significant inhibitory effect on 12 aquatic pathogenic bacteria, and the minimum inhibitory concentrations ranged from 20.6 to 61.7 μg/mL, basically reaching the same antibacterial effect as the antibiotic florfenicol. The survival rate of zebrafish embryos and CIK cells was almost 100% in the range of MIC. This study shows that V_C-CDs have good bactericidal and antibacterial effects on the main pathogenic bacteria in aquaculture, and have good biocompatibility, which signifies the potential as a promising alternative to antibiotics in the prevention and control of bacterial diseases in aquaculture.