The molecular chaperone Hsp90 is essential in eukaryotes, in which it facilitates the folding of developmental regulators and signal transduction proteins known as Hsp90 clients. In contrast, Hsp90 is not essential in bacteria, and a broad characterization of its molecular and organismal function is lacking. To enable such characterization, we used a genome-scale phylogenetic analysis to identify genes that co-evolve with bacterial Hsp90. We find that genes whose gain and loss were coordinated with Hsp90 throughout bacterial evolution tended to function in flagellar assembly, chemotaxis, and bacterial secretion, suggesting that Hsp90 may aid assembly of protein complexes. To add to the limited set of known bacterial Hsp90 clients, we further developed a statistical method to predict putative clients. We validated our predictions by demonstrating that the flagellar protein FliN and the chemotaxis kinase CheA behaved as Hsp90 clients in Escherichia coli, confirming the predicted role of Hsp90 in chemotaxis and flagellar assembly. Furthermore, normal Hsp90 function is important for wild-type motility and/or chemotaxis in E. coli. This novel function of bacterial Hsp90 agreed with our subsequent finding that Hsp90 is associated with a preference for multiple habitats and may therefore face a complex selection regime. Taken together, our results reveal previously unknown functions of bacterial Hsp90 and open avenues for future experimental exploration by implicating Hsp90 in the assembly of membrane protein complexes and adaptation to novel environments.