Heat shock protein 90 (HSP90), a molecular chaperone, has been identified as a drug target in inflammatory skin diseases. However, four different HSP90 isoforms (HSP90α, HSP90β, GRP94, TRAP1) exist. Therefore, this study aimed to evaluate the functional role of the HSP90 isoforms in skin inflammation. Selective knockdown of the HSP90 isoforms revealed different inflammatory effects in stimulated keratinocytes. TRAP1 knockdown significantly downregulated the expression of the measured inflammatory genes (IL1B, IL6, IL17C, IL23A, IL19, IL36G, CXCL8, CCL5, CCL17, CCL20). Selective and combined knockdown of HSP90α and HSP90β showed a trend towards increased inflammatory activity. Selective GRP94 knockdown and combined knockdown of the organelle-specific isoforms (GRP94+TRAP1) or all four isoforms resulted in inconsistent effects. Additionally, a selective-TRAP1 inhibitor (gamitrinib) suppressed the inflammatory gene expression in keratinocytes and fibroblasts (IL17C, IL23A, IL36G) and in hidradenitis suppurativa skin cultured ex vivo (IL1B, IL6, CXCL8, IL17A). In conclusion, selective and simultaneous knockdown of the HSP90 isoforms mediated different inflammatory effects, revealing that the HSP90 isoforms have distinct roles in skin inflammation. In addition, we discovered that inhibition of TRAP1 exerted consistent anti-inflammatory effects, suggesting that TRAP1 inhibitors may represent a topical therapeutic strategy for inflammatory skin diseases.