Streptococcus suis is a significant zoonotic agent affecting both human and pig health and poses a substantial public health concern. The pathogenicity of S. suis is intricately linked to its ability to form biofilms and express virulence factors, which are regulated by the LuxS/AI-2 quorum sensing (QS) system. Herein, we uncover a novel therapeutic avenue by demonstrating that 5-fluorouracil (5-FU), an FDA-approved anti-cancer agent, effectively mitigates biofilm formation and attenuates the virulence of S. suis. Mechanistically, we observe a significant reduction in capsular polysaccharide and extracellular polysaccharide production upon 5-FU treatment, elucidating a potential mechanism for biofilm weakening. Additionally, 5-FU down-regulates virulence traits, diminishing S. suis’s ability to adhere to host cells and evade phagocytosis. Crucially, our study identifies the thymidylate synthase regulatory gene thyA as a key mediator of 5-FU’s effects on the LuxS/AI-2 QS system. Virtual molecular docking and gene knockout experiments provide compelling evidence that 5-FU modulates the LuxS/AI-2 QS system by targeting thyA. In vivo experiments further validate the therapeutic potential of 5-FU, showcasing a significant reduction in bacterial load and mitigation of tissue damage in a mouse model. In conclusion, our investigation unveils 5-FU as a potent disruptor of S. suis’s biofilm formation and virulence, offering a promising avenue for the control of this devastating pathogen.