Targeting Heat shock protein 90 (HSP90) C-terminus is an important strategy to develop HSP90 inhibitors without inducing heat shock response. The development of C-terminal inhibitors, however, is hampered by a lack of understanding regarding the interaction between the HSP90 C-terminus and the present inhibitors. We collected seven classical and structurally diverse HSP90 C-terminal inhibitors and constructed a ligand-based pharmacophore model. The subsequent virtual screening and structural optimisation led to the identification of 2-heteroarylthio-N-arylacetamides as novel HSP90 C-terminal inhibitors. 9 and 27 exhibited strong antitumour activity in vitro by inhibiting proliferation and inducing apoptosis in multiple cancer cell lines. These compounds disrupted the interaction between HSP90 C-terminus and peptidylprolyl isomerase D, exerting a stronger inhibitory effect than novobiocin. 27 significantly induced the degradation of HSP90 clients without triggering heat shock response. In an in vivo study using 4T1 mice breast cancer models, 9 showed a potent antitumour effect without obvious toxicity.