Multidrug efflux is an obstacle to the successful treatment of infectious diseases, and it is mediated by multidrug efflux pumps that recognize and export a broad spectrum of chemically dissimilar toxic compounds. Many bacterial genome sequences have been determined, allowing us to identify drug efflux genes encoded in the bacterial genome. Here, we present an approach to identifying drug efflux genes and their regulatory networks in Escherichia coli and Salmonella. Multidrug efflux pumps are often regulated by environmental signals and they are required for bacterial virulence in addition to multidrug resistance. It is now understood that these efflux pumps also have physiological roles. In this article, we investigate the physiological roles of drug efflux pumps in virulence. Because multidrug efflux pumps have roles in bacterial drug resistance and virulence, we propose that drug efflux pumps have greater clinical relevance than previously considered.