Glioblastoma (GBM) is an aggressive and highly malignant primary brain tumour, accounting for a significant proportion of adult brain tumours. It is associated with a poor prognosis and high recurrence rates. Although temozolomide (TMZ) remains the standard first-line chemotherapy for GBM, its clinical efficacy is often limited by the development of drug resistance and toxic effects on normal tissues. Melatonin (Mel), a natural indoleamine synthesised by the pineal gland, has demonstrated synergistic anti-tumour effects when combined with various chemotherapy agents in multiple studies. This study investigates the synergistic potential of Mel to enhance TMZ's therapeutic efficacy against GBM. The results demonstrate that the combination of Mel and TMZ significantly inhibits glioblastoma cell proliferation, migration, and invasion. Mechanistically, this synergistic effect is mediated through the NF-κB/COX-2 signalling pathway. Mel enhances TMZ's anti-tumour activity by inhibiting IκBα phosphorylation, suppressing NF-κB activation, and downregulating COX-2 expression. Additionally, the combination treatment induced apoptosis via activation of the Caspase-3 pathway. These results suggest that Mel can potentiate the therapeutic efficacy of TMZ in glioblastoma treatment, offering a promising strategy to overcome TMZ resistance while reducing its associated toxicity.