Glioblastoma (GBM) remains a formidable clinical challenge, with cancer stem cells (CSCs) contributing to treatment resistance and tumor recurrence. Conventional treatments often fail to eradicate these CSCs characterized by enhanced resistance to standard therapies through metabolic plasticity making them key targets for novel treatment approaches. Addressing this challenge, this study introduces a novel combination therapy of dichloroacetate (DCA), a metabolic modulator and nonthermal plasma to induce oxidative stress in glioblastomas. Our results demonstrate that DCA and nonthermal plasma (NTP) synergistically increase ROS production, resulting in endoplasmic reticulum (ER) stress and mitochondrial reprogramming, key factors in the initiation of programmed cell death. Furthermore, the combination downregulated key stemness markers indicating effective CSCs suppression. Upregulation of pro-apoptotic proteins and downregulation of anti-apoptotic factors highlight the induction of apoptosis in glioma stem cells. This study provides compelling evidence that the combination of DCA and NTP offers a novel and effective strategy for targeting glioma CSCs by inducing oxidative and metabolic stress, underscoring potential therapeutic advancements in glioblastoma treatment.