Glioblastoma Multiforme (GBM) is an aggressive brain tumor with a poor prognosis, highlighting the need for more effective therapeutic strategies. One promising approach involves telomerase inhibition, as telomerase is essential for cellular immortality by maintaining telomere length. Furthermore, the discovery of telomerase reverse transcriptase (TERT) promoter mutations revealed the most common oncogenic mutation in glioblastoma. Non-nucleoside reverse transcriptase inhibitors (NNRTIs) have shown potential as telomerase inhibitors, leveraging the structural similarities between telomerase and retroviral reverse transcriptase. Methods: This study explores the therapeutic potential of two novel Human T-lymphotropic virus ۱ (HTLV-۱) reverse transcriptase (RT) inhibitors, compounds ۱۰۱ and ۱۰۲, for treating GBM. Initially, an in-silico evaluation of the compounds was performed using homology modeling and molecular docking approaches. The binding affinities of the compounds, relative to BIBR۱۵۳۲, were assessed through computational analysis. Their effects were studied using T۹۸G and U۲۵۱ GBM cell lines through various assays, including viability assays, real-time polymerase chain reaction (qRT-PCR), Propidium iodide (PI) staining, Annexin PI double staining, and apoptotic gene expression analysis. Their efficacy was also compared to BIBR۱۵۳۲, a known telomerase inhibitor. Results: The molecular docking simulations revealed that both compounds ۱۰۱ and ۱۰۲ had similar or better binding affinities for the human telomerase active site compared to BIBR۱۵۳۲. Follow-up telomere length assays demonstrated that these compounds significantly reduced telomere length. Further analysis showed that these compounds induced specific cell cycle phase arrests. Compound ۱۰۱ predominantly triggered G۱ arrest, while compound ۱۰۲ and BIBR۱۵۳۲ caused significant G۲/M arrest. Importantly, compound ۱۰۲ exhibited stronger effects than BIBR۱۵۳۲, as it was more effective in inhibiting cell proliferation and inducing apoptosis. Conclusion: In conclusion, compounds ۱۰۱ and ۱۰۲ demonstrate strong potential as telomerase inhibitors with therapeutic applications for GBM. Their higher potency compared to BIBR۱۵۳۲ suggests that they could be developed further as viable treatment options. Additional preclinical studies are recommended to fully evaluate their therapeutic potential in combating GBM and improving patient outcomes.