Heat shock protein 90 (Hsp90) is a molecular chaperone essential for the stability and function of multiple cellular client proteins, a number of which have been implicated in the pathogenesis of breast cancer. Here we undertook a comprehensive evaluation of the activity of ganetespib, a selective Hsp90 inhibitor, in this malignancy. With low nanomolar potency, ganetespib reduced cell viability in a panel of hormone receptor-positive, HER2-overexpressing, triple-negative and inflammatory breast cancer cell lines in vitro. Ganetespib treatment induced a rapid and sustained destabilization of multiple client proteins and oncogenic signaling pathways and even brief exposure was sufficient to induce and maintain suppression of HER2 levels in cells driven by this receptor. Indeed, HER2-overexpressing BT-474 cells were comparatively more sensitive to ganetespib than the dual HER2/EGFR tyrosine kinase inhibitor lapatinib in three-dimensional culture. Ganetespib exposure caused pleiotropic effects in the inflammatory breast cancer line SUM149, including receptor tyrosine kinases, MAPK, AKT and mTOR signaling, transcription factors and proteins involved in cell cycle, stress and apoptotic regulation, as well as providing combinatorial benefit with lapatinib in these cells. This multimodal activity translated to potent antitumor efficacy in vivo, suppressing tumor growth in MCF-7 and MDA-MB-231 xenografts and inducing tumor regression in the BT-474 model. Thus, ganetespib potently inhibits Hsp90 leading to the degradation of multiple clinically-validated oncogenic client proteins in breast cancer cells, encompassing the broad spectrum of molecularly-defined subtypes. This preclinical activity profile suggests that ganetespib may offer considerable promise as a new therapeutic candidate for patients with advanced breast cancers.