Molecular chaperones, as the name suggests, are involved in folding, maintenance, intracellular transport and degradation of proteins as well as in facilitating cell signaling. Heat-shock protein 90 (Hsp90) is an essential eukaryotic molecular chaperone that carries out these processes in normal and cancer cells. Hsp90 function in vivo is coupled to its ability to hydrolyze ATP and this can be regulated by co-chaperones and post-translational modifications. In this review, we explore the varied roles of known post-translational modifications of Hsp90 (phosphorylation, acetylation, S-nitrosylation, oxidation and ubiquitination) in fine-tuning chaperone function in eukaryotes. We also provide examples of Hsp90 inhibitor-based clinical activity in several molecularly distinct cancers and we use these examples to suggest how and in what cancers Hsp90 inhibitors are most likely to be effective, either as single agents or in combination with additional therapies.