The Burkholderia cepacia complex (Bcc) are multi-drug resistant, opportunistic pathogens that cause infections in immunocompromised patients and those with cystic fibrosis. A chemogenomic screen of a transposon mutant library of essential genes revealed a novel two-component system, named esaSR that affects the antimicrobial resistance of Burkholderia cenocepacia. To characterize esaSR, an esaS knockout mutant and esaR knockdown mutant were created. To characterize the phenotype of resultant mutants, viability, minimum inhibitory concentrations, RND efflux expression, membrane protein profiles were assessed. Results demonstrate that esaR is essential for growth and viability in rich media. Mutants also exhibit hypersusceptibility to antimicrobials, altered cell size and morphology, altered RND efflux expression, in addition to having compromised cell envelopes. In summary, results indicate that EsaSR plays a major role in the viability and antimicrobial resistance of B. cenocepacia. EsaSR represents an attractive system to study for its potential as a drug target to lower resistance and render resistant pathogens susceptible to antibiotics currently in use.