Hsp90 is a target for anti-cancer drug development. Both the conformational events tuned by ATP/ADP and co-chaperones and the chaperoning cycle timing are required for Hsp90's fully functional display. Interfering with either one of the conformational events or the cycle timing will down-regulate Hsp90's function. In this manuscript, non-covalent allosteric modulators (SOMCL-16-171 and SOMCL-16-175) targeting Hsp90α's middle domain (Hsp90M) were developed for the first time. Multiple techniques were then applied to characterize the interactions between two active compounds and Hsp90α. Two loops and one α-helix (F349-N360, K443-E451, and D372-G387) in Hsp90M were identified responsible for the recognition of SOMCL-16-171 and SOMCL-16-175. Meanwhile, the binding of SOMCL-16-171 and SOMCL-16-175 to Hsp90M was demonstrated to allosterically modulate the structure and function of Hsp90α's N-terminal domain. Finally, cellular assays were conducted to evaluate the cellular activity of SOMCL-16-175, and the results indicate that SOMCL-16-175 destabilizes Hsp90's client proteins and reduces cell viability.