© 2021 Elsevier GmbHIntroduction: Glioblastoma (GB) is the most aggressive primary brain tumour. Temozolomide (TMZ) is a chemotherapy drug used in the treatment of GB. Despite treatment with TMZ, the prognosis of GB is poor. This study aimed to demonstrate the ability of Olea europaea leaf extract (OLE) alone and in combination with TMZ to suppress tumour aggressiveness by evaluating long non-coding RNA (LncRNA) and cancer stem cell (CSC) markers in GB cells using a three-dimensional (3D) model. Methods: The Real-time PCR (RT-PCR) method was used to determine the effects of OLE on LncRNA and CSC markers associated with tumour aggressiveness. To explore the effect of OLE on tumour size, a 3D model was developed. Results: It was found that OLE suppressed tumour aggressiveness with inhibited the MALAT1, SOX2 and NANOG (p < 0.05). OLE+TMZ also inhibited MALAT1, LOXL1-AS1, PVT1 and H19 (p < 0.05) and OCT4, NANOG, SOX2 and CD133 (p < 0.05). In addition, to reduce tumour aggressiveness in a 3D cell culture, the use of OLE and OLE+TMZ has been supported (47.11-fold, p < 0.0001 and 18.04-fold, p < 0.0001, respectively). Conclusion: OLE may be a potential therapeutic agent that can be used in the treatment of GB, as it has been shown to reduce tumour size and increase the effect of TMZ.