<p>Biofilm formation in <i>Burkholderia thailandensis</i>, a model for the causative agent of melioidosis, is critically regulated by <i>N</i>-acyl homoserine lactone (AHL)-mediated quorum sensing (QS), contributing to its pathogenicity and treatment failure. This study investigates a novel combinatorial strategy to disrupt this process by employing methyl gallate, a plant-derived biofilm inhibitor, together with the AHL-lactonase YtnP. Our results demonstrate that methyl gallate not only attenuated <i>B. thailandensis</i> virulence in a <i>Galleria mellonella</i> infection model but also showed a markedly slower development of resistance compared to conventional antibiotics like tetracycline and meropenem. Strikingly, the combination of sub-inhibitory concentrations of methyl gallate and YtnP exhibited a synergistic effect, significantly enhancing the susceptibility of the bacterium to methyl gallate. This synergy was underpinned by a profound suppression of AHL signal molecules (C8-HSL, 3-OH-C8-HSL, and 3-OH-C10-HSL) and a concurrent downregulation of key QS regulatory genes (<i>btaI1-3</i>, <i>btaR1-3</i>). Consequently, the combination treatment robustly impaired biofilm formation and disrupted its structural integrity, as visualized by confocal microscopy. Molecular docking analyses provided a structural basis for this synergy, revealing stable binding of C8-HSL within the catalytic pocket of YtnP. Our findings posit the methyl gallate-YtnP combination as a potent and resistance-retarding anti-virulence approach against <i>B. thailandensis</i> biofilms.</p>