Gliomas are the most common malignant brain tumors in adults, characterized by a high proliferation and invasiveness. Treatment remains challenging due to their immunomodulatory properties, which promote immune evasion and tumor progression. Given the substantial consumption of yerba mate in the southern hemisphere, this study evaluated the effects of caffeinated and decaffeinated yerba mate infusion extracts (ECaf and EDCaf, respectively) on glioblastoma cell lines (U87 and U251). Both extracts reduced cell viability in a dose-dependent manner, with U87 being more sensitive. ECaf and EDCaf inhibited cell migration and adhesion, particularly in U87 cells. Real-time PCR showed a reduction in CD73 and MMP2 expression in U87, whereas U251 exhibited a slight increase in MMP2 expression. CD73 enzymatic activity was reduced in U87 cells by both extracts but remained unaffected in U251 cells. Overall, the results suggest that caffeine absence does not alter the bioactivity of yerba mate extracts, highlighting their potential to modulate glioblastoma cell behavior.