Temozolomide (Tmz) is a DNA methylating agent used for the treatment of glioblastoma multiforme (GBM). Resistance to Tmz in GBM is caused by the DNA direct repair enzyme O6-methylguanine DNA methyltransferase (MGMT), which is expressed in ~50% of GBM tumours. It has yet to be confirmed that MGMT acts within mitochondria to repair mitochondrial DNA (mtDNA), and in this report we discuss the development of a novel mitochondria-targeted temozolomide probe (mtTmz) for evading MGMT-mediated resistance. Through conjugation of Tmz to a mitochondria-penetrating peptide (MPP), exclusive mitochondrial localization was achieved, and the probe retained alkylation activity demonstrated by chemical and DNA-based assays. Absence of nuclear DNA damage was assessed by detecting γH2AX foci. mtTmz demonstrated efficient cell killing capabilities independent of MGMT status in GBM cells as determined by cell viability assays. It was determined using a Proteinase K digestion assay that MGMT does not translocate to mitochondria in response to mtTmz treatment, and RT-qPCR analysis demonstrated that mtTmz does not induce MGMT gene expression compared to Tmz. The results reported highlight both the potential of mitochondrial targeting of Tmz and mitochondria as a therapeutic target in MGMT-expressing GBM.