In spite of the many studies carried out on glioblastoma multiforme (GBM) in recent years and the implementation of promising new therapeutic strategies, median survival rates in GBM patients have not been improved. A key issue in the fight against a tumor is learning its biology which is only possible in laboratory conditions that accurately reproduce its physiological microenvironment. GBM is among the most hypoxic and glycolytic tumors of the central nervous system. The most recent data suggest that these two metabolic features should be recognized as the most important markers of the GBM aggressive phenotype associated with its resistance to chemo- and radiotherapy. However, until now, the effect of modulation of the tumor’s microenvironment on drug efficacy has been evaluated only in a few studies. Since hypoxia inside a tumor creates protection for GBM cells from chemotherapy and the spatial arrangement of hypoxia in GBM is a dynamic process (the level of pO2 inside a tumor is from 0% to 5%), our experiments performed on the T98G glioblastoma cell line were carried out in a range of oxygen availability conditions and then compared to standard laboratory conditions. Moreover, the influence of high or standard glucose concentration or its deprivation in the culture medium on temozolomide effect (the standard chemotherapeutic for GBM) was estimated. We examined the influence of temozolomide on cell viability, division and apoptosis as well as on migration of tumor cells by using the time-lapse real-time observations. Our study showed that glioblastoma cells cultured in conditions of chronic hypoxia were almost completely resistant to the effect of the drug. Moreover, we observed that increased glucose availability in the GBM microenvironment was associated with the enhanced resistance of the GBM to the cytotoxic effect of temozolomide in hypoxic conditions which was manifested by an increased cellular viability and division and a diminished percentage of apoptotic cells.