Glycyrrhizin (GLR), an active constituent of the traditional Chinese medicine (TCM) liquorice (Glycyrrhiza uralensis Fisch), has important functions against bacterial invasions in mammals. However, studies on the antimicrobial properties of GLR in teleosts remain relatively scarce. Herein, we performed an in vitro screening of 30 monomeric TCM compounds and found that only GLR exhibited significant inhibitory activity against the bacterium Pseudomonas plecoglossicida and increased the survival rate of ayu fish challenged with it. Moreover, we explored the antimicrobial properties of GLR in in vitro antimicrobial experiments and found that it inhibited all 10 common aquatic pathogens, including P. plecoglossicida. Notably, spectrophotometry and fluorescence staining assays revealed that GLR exhibited bactericidal activity in vitro by inhibiting P. plecoglossicida biofilm formation and increasing its membrane permeability. Notably, we showed that GLR could reduce pro-inflammatory factor levels, reduce bacterium-triggered apoptosis of monocytes/macrophages (MO/Mф), and promote their phagocytosis and bactericidal capacity. Mechanically, a cellular thermal shift assay confirmed that GLR could bind to high mobility group box 1 (HMGB1), suggesting that GLR may regulate MO/Mф functions through HMGB1-mediated pathways. Overall, our findings confirm the direct bactericidal ability of GLR and resistance to pathogen invasion by modulating the immune activity of MO/Mф, providing an essential basis for the utilisation of TCM in aquatic animal protection.