Abstract: Burkholderia pseudomallei (Bp) and Burkholderia mallei (Bm) are Tier-1 select pathogens that cause highly lethal human infections with limited therapeutic options. Intercellular spread is a hallmark of Burkholderia pathogenesis and its prominent ties to virulence make it an attractive therapeutic target. We developed a high-throughput cell-based phenotypic assay and screened ˜220,000 small molecules for their ability to disrupt intercellular spread by Burkholderia thailandensis, a BSL-2 surrogate for these pathogens. 268 hits were identified, and cross-species validation found 32 hits that also disrupt intercellular spread by Bp and/or Bm. In a fulminant murine model of respiratory melioidosis, treatment with a number of these agents was significantly more effective than ceftazidime, the current drug of choice, for improving patient survival and decreasing bacterial counts in major organs.