Burkholderia pseudomallei exists as a normal resident of tropical and sub-tropical soils worldwide, often infecting hosts via incidental exposure to disturbed soils in the wake of natural disasters and severe weather events. The resulting disease Melioidosis constitutes a life-threatening challenge, as B. pseudomallei aggressively colonises most host tissues contributing to difficulties in clinician recognition and treatment efficacy. Understanding what supports B. pseudomallei expansion within the host could provide new targets for anti-virulence drug design. The enzyme effector trehalase (treA) is used by B. pseudomallei for the hydrolysis of the disaccharide trehalose, a molecule found in the environment and host tissues. Interestingly, trehalose metabolism has been demonstrated to significantly impact virulence in multiple melioidosis host models1. This work aims to further define the participation of treA in both external survival and virulence, with the purview that novel inhibitors targeted against treA could assist therapeutic intervention. To re-establish the phenotype demonstrated in prior studies, a new unmarked deletion mutant (ΔtreA) was constructed in our laboratory B. pseudomallei K96243. This mutant was assessed for its capacity to replicate wild-type B. pseudomallei infectivity within an in vitro macrophage model, in addition to static biofilm formation, and growth on trehalose. In comparison to wild-type B. pseudomallei, ΔtreA was unable to grow when provided with trehalose as the sole carbon source (p<0.0001). This was fully restored upon complementation with B. pseudomallei K96243 treA. Furthermore, ΔtreA exhibited significantly diminished biofilm formation capacity (p<0.001) after 72-hours of continuous growth. Using novel trehalase inhibitors, we now seek to replicate these in vitro effects with concurrent investigation into the localization of trehalase protein within the B. pseudomallei microenvironment, via tagged complementation. Our preliminary investigations reveal the potential utility of pursuing trehalase as a novel target for therapeutic intervention. By interfering with trehalase, prospective inhibitors may constrain the rapid growth of B. pseudomallei in patients and reduce the outright virulence permitted by normal metabolic activity.