Structural analysis of tazemetostat, an FDA-approved EZH2 inhibitor, led us to pinpoint a suitable site for appendage with a pharmacophoric fragment of second-generation HSP90 inhibitors. Resultantly, a magnificent dual EZH2/HSP90 inhibitor was pinpointed that exerted striking cell growth inhibitory efficacy against TMZ-resistant Glioblastoma (GBM) cell lines. Exhaustive explorations of chemical probe led to several revelations such as (i) compound increased apoptosis/necrosis-related gene expression, whereas decreased M phase/kinetochore/spindle-related gene expression as well as CENPs protein expression in Pt3R cells; (ii) dual inhibitor induced cell cycle arrest at the M phase; (iii) compound suppressed reactive oxygen species (ROS) catabolism pathway, causing the death of TMZ-resistant GBM cells; and (iv) compound elicited substantial anti-GBM efficacy in experimental mice xenografted with TMZ-resistant Pt3R cells. Collectively, the study results confirm the potential of dual EZH2-HSP90 inhibitor as a tractable anti-GBM agent.