Pediatric high-grade gliomas (pedHGGs), including diffuse midline gliomas (DMGs), are the most aggressive tumors with the poorest overall survival amongst infant population. This underscores the urgent necessity of novel, alternative therapies. The oncolytic adenovirus Delta-24-RGD and the imipridone ONC201 are two of the most promising therapeutic agents in the field, having demonstrated safety and effectiveness in preclinical and clinical settings. Therefore, we aimed to evaluate whether the Delta-24-RGD/ONC201 combination could result in an increased therapeutic benefit in pedHGGs and DMGs. Given that ONC201 targets mitochondrial metabolism, we assessed potential negative interactions of the combination therapy. We found that ONC201 cotreatment with Delta-24-RGD did not affect in the virus replication capability in vitro. Interestingly, citotoxycity analyses showed that cotreatment is either synergistic (4/6) and additive (2/6) in a panel of human pedHGG and DMG cell lines. Mechanistic analyses showed a sustained decrease in oxygen consumption rate and a reduction of mitochondrial DNA; an increase of DMA damage measured by pH2A.X; and an increase of endoplasmic reticulum stress via PERK-ATF4-CHOP signaling in combination treatment. Altogether, these data suggest an improved response to the combination via mitochondria and endoplasmic reticulum perturbation. Notably, Delta-24-RGD/ONC201 cotreatment in human xenograph-bearing mice of pedHGG and DMG depicted a significant improvement over each agent alone. Encouragingly, a reshaping of the tumor microenvironment in an immunocompetent model towards a proinflammatory phenotype was revealed, which produced the increase of lymphoid (CD4+, CD8+, Treg and NK cells) and myeloid (macrophages, granulocytes, monocytes, denditric cells and microglia) linage.