Significance Multidrug resistance (MDR) constitutes a major obstacle in the treatment of infectious diseases, as well as human cancers, even in developed countries. This study deciphers the structural mechanism that dictates the promiscuous multidrug binding to the variable transcriptional regulation of QacR, the major multidrug-binding transcriptional repressor in pathogenic bacteria Staphylococcus aureus . The transcriptional level of the downstream transporter gene, qacA , is dynamically regulated by QacR via the relative populations of inducive versus repressive conformations in the molecule. The conformational equilibrium also defines the basal activity of the MDR system, as well as the elevated transcription levels with the constitutively active mutants. Our structural definition provides an important advance in understanding and potentially targeting the MDR system.