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The Tier 1 Select Agent Burkholderia pseudomallei is an intrinsically antibiotic resistant bacteria that is virulent in airways, and naturally abundant, deeming it a threat to public health. New information on the virulent capabilities of B. thailandensis, a safer, validated surrogate for B. pseudomallei, has recently been uncovered, however, a global metabolomic analysis of the infection in vitro is lacking. To address this gap, dual metabolomic profiling was applied to two cell types known to interact with B. thailandensis during infection using an untargeted LC-MS/MS analysis. Here we report the detection of several key B. thailandensis-produced secondary metabolites in vitro in B. thailandensis-infected mouse cell lines as well as the depletion of lysophosphatidylcholines during infection. We also demonstrate that there are distinct metabolomic profiles attributed to different bacterial loads and infection lengths in vitro due to the presence of unique metabolites under each condition. Our results provide new insight into the chemical interactions between B. thailandensis and its hosts, and implicate the role of distinct metabolites in B. thailandensis pathogenesis.