Objective This study employed the two-sample mendelian randomization (TSMR) method to investigate the potential causal relationship between intestinal flora and glioblastoma (GBM).Methods The research utilized Mendelian randomization (MR) approach to identify single nucleotide polymorphisms (SNPs) associated with the onset of brain glioblastoma from 211 large-scale genome-wide association studies (GWAS) related to intestinal microbiota. SNPs were used as instrumental variables. The inverse variance weighting (IVW) method was primarily applied to assess the causal effect, while heterogeneity testing, pleiotropy assessment, and sensitivity analysis were performed to ensure the reliability of the findings. Furthermore, the study excluded SNPs with horizontal pleiotropy outliers, thereby enhancing the validity of the conclusions. Results Mendelian randomization analysis revealed potential associations between nine gut microbiota species and glioblastoma (GBM). Specifically, an increased risk of GBM was observed in the presence of Eubacterium brachygroup and Peptostreptococcaceae, while Ruminococcaceae, Prevotella, Faecalibacterium, Phascolarctobacterium, Anaerostipes, Lachnospiraceae, and Streptococcus were associated with a decreased risk of GBM. These nine microbial species demonstrated stability in terms of heterogeneity and horizontal pleiotropy. Furthermore, after rigorous BH correction, Ruminococcaceae was found to exhibit a protective effect against GBM (OR IVW=0.036, 95%CI:0.007-0.191, PBH=0.003).Conclusion This study demonstrates that the taxa Ruminococcaceae and Prevotella exert a protective role against the occurrence and development of glioblastoma (GBM), whereas the genera Eubacteriumbrachygroup and Peptostreptococcaceae are potential risk factors for GBM. Modulating the gut microbiota presents promising clinical implications for the prevention and treatment of GBM