CUDC-907, referred to as Fimepinostat, functions as a dual inhibitor of PI3K and HDAC, exhibiting significant anti-tumor efficacy in a range of cancer types. However, its specific role in glioblastoma is not well understood. In this study, we investigated the effects of CUDC-907 on glioblastoma using cellular, organoid, and animal models to assess its inhibitory potential and toxicity. In vitro, we assessed glioblastoma cell proliferation, migration, invasion, and apoptosis using standard assays. glioblastoma organoids were treated to examine three-dimensional tumor growth and cellular changes. For in vivo analysis, animal models with glioblastoma received CUDC-907 to study its systemic impact and toxicity, with tumor progression closely monitored. We also tested the synergy between CUDC-907 and temozolomide to evaluate enhanced chemosensitivity. RNA sequencing was conducted to explore the fundamental molecular mechanisms involved, focusing on alterations in the cell cycle and DNA damage repair pathways. Our findings show that CUDC-907 significantly suppresses the proliferation, migration, and invasion of glioblastoma cells and promotes apoptosis, while exhibiting minimal toxicity. Additionally, CUDC-907 acts synergistically with temozolomide, a chemotherapy drug used for glioblastoma treatment, enhancing glioblastoma’s chemosensitivity to temozolomide. RNA sequencing suggests that CUDC-907 achieves its effects by influencing the glioblastoma cell cycle and inhibiting DNA damage repair. Overall, the data suggest that CUDC-907 may be a promising anti-cancer agent for glioblastoma treatment.