This study aims to elucidate the potential mechanisms underlying the therapeutic effects of Bletilla striata in glioma. Targets of Bletilla striata and glioma were predicted using TCMSP, SwissTargetPrediction, GeneCards, and other databases. A "drug-ingredient-target" network and protein-protein interaction (PPI) network were constructed, with core targets identified via topological analysis. Functional enrichment (GO/KEGG, DAVID), molecular docking, and experimental validations (MTT, scratch assay, RT-PCR, Western blot) were performed. A total of 11 active ingredients of Bletilla striata, 456 corresponding targets, and 2,830 glioma-related targets were identified. Nine core targets (AKT1, STAT3, mTOR, etc.) were identified. GO analysis indicated that these targets were primarily involved in phosphorylation, protein binding, and the positive regulation of RNA polymerase II transcription. KEGG analysis highlighted key pathways, including pathways in cancer, the PI3K-AKT signaling pathway, and microRNA-related regulatory mechanisms in cancer. Molecular docking analysis demonstrated high binding affinities between active ingredients and core targets, particularly AKT1 and mTOR. Functional assays showed that Blestriarene A, a key active compound in Bletilla striata, significantly suppressed glioma cell proliferation and migration. RT-PCR results indicated that treatment with varying concentrations of Blestriarene A for 48 h downregulated AKT mRNA expression while upregulating mTOR mRNA expression. Western blot analysis further confirmed a reduction in PI3K, AKT, and mTOR phosphorylation following treatment. Network pharmacology and in vitro experiments suggest that Blestriarene A anti-glioma effects by modulating the PI3K/AKT/mTOR signaling pathway.