For a study, researchers sought to test Gamitrinib, a mitochondrial-localized HSP90 inhibitor, for antitumor activity in multiple glioma models to elucidate Gamitrinib’s antitumor mechanisms in gliomas. To investigate Gamitrinib’s therapeutic efficacy, a large panel of primary and temozolomide (TMZ)-resistant human glioma cell lines was screened using cell viability assays, flow cytometry, and crystal violet assays. The mitochondrial respiration of glioma cells was measured using seahorse assays. Gamitrinib’s potential antitumor mechanisms were discovered using integrated analyses of RNA sequencing (RNAseq) and reverse-phase protein array (RPPA) data. To further evaluate Gamitrinib’s therapeutic efficacy, neurospheres, patient-derived organoids (PDO), cell line-derived xenografts (CDX), and patient-derived xenografts (PDX) models were created. In 17 primary glioma cell lines, 6 TMZ-resistant glioma cell lines, 4 neurospheres, and 3 PDOs, gefitinib inhibited cell proliferation and induced apoptosis and death. In addition, Gamitrinib significantly delayed tumor growth and improved survival in both CDX and PDX models in which tumors were implanted subcutaneously or intracranially. Gamitrinib exhibited antitumor activity by suppressing mitochondrial biogenesis, OXPHOS, and cell-cycle progression and activating the energy-sensing AMP-activated kinase, DNA damage, and stress response, according to integrated computational analyses of RNAseq and RPPA data. These preclinical findings established Gamitrinib’s therapeutic role in gliomas and revealed that the primary antitumor mechanisms in gliomas were the inhibition of mitochondrial biogenesis and tumor bioenergetics.