Aeromonas hydrophila, a Gram-negative bacterium, poses significant threats to aquaculture, leading to substantial economic losses. Its pathogenicity is primarily driven by a sophisticated quorum sensing (QS) system that regulates virulence factors. This study investigates saponins extracted from the sea cucumber Holothuria leucospilota as potential natural inhibitors of QS in A. hydrophila, offering a novel disease management strategy for aquaculture. Specimens of H. leucospilota were collected, and saponins were extracted from their Cuvierian tubules through a process of homogenization, solvent extraction, and purification. The saponin extract's minimum bactericidal concentration (MBC) and minimum inhibitory concentration (MIC) against A. hydrophila were found to be 80 μg/mL and 30 μg/mL, respectively. Hemolysin, lipase, and protease are examples of QS-regulated virulence factors whose activities were found to be significantly reduced by sub-MIC levels of saponins. Additionally, swarming motility and biofilm formation were notably inhibited. A significant downregulation of the QS genes ahyI and ahyR was observed, indicating an effective disruption of the QS system.These findings suggest that saponins from H. leucospilota can inhibit the QS system in A. hydrophila, thereby reducing its pathogenicity. This disruption offers a promising method for controlling bacterial infections without directly killing the bacteria, potentially mitigating antibiotic resistance. The study highlights the potential of marine-derived compounds as natural QS inhibitors, contributing to environmentally friendly aquaculture practices. Overall, it provides evidence that sea cucumber saponins could serve as a novel class of anti-QS agents, presenting a new perspective on disease management in aquaculture and other bacterial infection contexts.