Gliomas present a formidable challenge for translational progress. Heterogeneity within and between tumors may demand empirically individualized insights, though relatively little is known about the biochemical milieu within which malignant cells thrive in the in vivo human glioma. We performed a pilot study of intraoperative high molecular weight microdialysis to sample the extracellular tumor environment within three locations in each of five molecularly diverse human gliomas spanning WHO grade 2 oligodendroglioma to WHO grade 4 glioblastoma (GBM). Microdialysates were subjected to targeted (D/L-2-hydroxyglutarate (2-HG)) and untargeted metabolomic analyses, enabling correlation, clustering, fold change, and enrichment analyses. IDH-mutant tumor microdialysate contained markedly higher levels of D2-HG than IDH-wild type tumors. However, IDH status was not predictive of the global metabolomic signature. Rather, two distinct metabolic phenotypes (α and β) emerged, with IDH-WT and IDH-mutant patient samples in each group. Individualized metabolic signatures of enhancing tumor versus adjacent brain were conserved across patients with glioblastoma regardless of metabolic phenotype. Untargeted metabolomic analysis additionally enabled correlative quantification of multiple peri-operatively administered drugs, illustrating regional heterogeneity of blood-brain barrier permeability. As such, acute intraoperative microdialysis affords a previously unharnessed window into individualized heterogeneous microenvironments within and between live human gliomas. Such access to the interstitial milieu of live human gliomas may provide a complementary tool for the development of individualized glioma therapies.