Glioblastoma (GBM) is the most common primary brain tumor. Tumor hypoxia is a pivotal factor responsible for the progression of this malignant glioma, and its resistance to radiation and chemotherapy. Thus, improved tumor tissue oxygenation may promote greater sensitivity to anticancer treatment. Protein kinase D1 (PKD1) protects cells from oxidative stress, and its abnormal activity serves an important role in multiple malignancies. The present study examined the effects of various oxygen conditions on the cytotoxic potential of the novel isothiourea derivate N,N'‑dimethyl‑S‑(2,3,4,5,6‑pentabromobenzyl)‑isothiouronium bromide (ZKK‑3) against the T98G GBM cell line. ZKK‑3 was applied at concentrations of 10, 25 and 50 µM, and cells were maintained under conditions of normoxia, anoxia, hypoxia, hyperbaric oxygen (HBO), hypoxia/hypoxia and hypoxia/HBO. The proliferation and viability of neoplastic cells, and protein expression levels of hypoxia‑inducible factor 1α (HIF‑1α), PKD1, phosphorylated (p)PKD1 (Ser 916) and pPKD1 (Ser 744/748) kinases were evaluated. Oxygen deficiency, particularly regarding hypoxia, could diminish the cytotoxic effect of ZKK‑3 at 25 and 50 µM and improve T98G cell survival compared with normoxia. HBO significantly reduced cell proliferation and increased T98G cell sensitivity to ZKK‑3 when compared with normoxia. HIF‑1α expression levels were increased under hypoxia compared with normoxia and decreased under HBO compared with hypoxia/hypoxia at 0, 10 and 50 µM ZKK‑3, suggesting that HBO improved oxygenation of the cells. ZKK‑3 exhibited inhibitory activity against pPKD1 (Ser 916) kinase; however, the examined oxygen conditions did not appear to significantly influence the expression of this phosphorylated form in cells treated with the tested compound. Regarding pPKD1 (Ser 744/748), a significant difference in expression was observed only for cells treated with 10 µM ZKK‑3 and hypoxia/hypoxia compared with normoxia. However, there were significant differences in the expression levels of both phosphorylated forms of PKD1 under different oxygen conditions in the controls. In conclusion, the combination of isothiourea derivatives and hyperbaric oxygenation appears to be a promising therapeutic approach for malignant glioma treatment.