Glioblastomas (GBMs) are notoriously difficult to treat, and the development of multiple drug resistance (MDR) is common during the course of the disease. The polyunsaturated fatty acids (PUFAs) gamma-linolenic acid (GLA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) have been reported to improve MDR in several tumors including breast, bladder, and leukaemia. However, the effects of PUFAs on GBM cell MDR are poorly understood. The present study investigated the effects of PUFAs on cellular responses to temozolomide (TMZ) in U87MG cells and the TMZ-resistant (TMZR) cells derived from U87MG. Cells were treated with PUFAs in the absence or presence of TMZ and dose–response, viable cell counting, gene expression, Western blotting, flow cytometry, gas chromatography-mass spectrometry (GCMS), and drug efflux studies were performed. The development of TMZ resistance caused an increase in ABC transporter ABCB1 and ABCC1 expression. GLA-, EPA-, and DHA-treated cells had altered fatty acid composition and accumulated lipid droplets in the cytoplasm. The most significant reduction in cell growth was seen for the U87MG and TMZR cells in the presence of EPA. GLA and EPA caused more significant effects on ABC transporter expression than DHA. GLA and EPA in combination with TMZ caused significant reductions in rhodamine 123 efflux from U87MG cells but not from TMZR cells. Overall, these findings support the notion that PUFAs can modulate ABC transporters in GBM cells.