Abstract Emerging molecular data demonstrates the importance of genomic and epigenetic factors in pathogenesis of meningioma. Understanding the metabolic landscape using mass spectrometry is needed to overcome significant uncertainty in predicting tumor behavior and the risk of recurrence. Current technologies lack sensitivity and selectivity, which hinders the discovery of potential novel diagnostic and prognostic features. Here, we present a nontargeted metabolomics approach applied to meningiomas for biomarker discovery and identification of potential therapeutic targets. Fresh frozen tissue from 36 patients (57% women, 43% men; mean age: 48) who underwent surgical resection for newly diagnosed meningiomas and 9 patient samples with non-neoplastic dura were used for case/control comparison. Additionally, 16 patient-derived meningioma cell lines (Grade I-III), 2 immortalized human cell lines (IOMM-Lee and Ch157MN) and immortalized arachnoid cells were used to further explore metabolic changes in meningiomas. Metabolites were extracted from tissue and cells for GC-TOF (primary metabolism), RPLC ESI (±) (lipidomics) and HILIC ESI (±) (biogenic amines) coupled to high-resolution mass spectrometry for analysis. Metabolites were identified using authentic standards, retention time, and MS2 fragmentation. Over one thousand known metabolites were identified and annotated as well as over 300 unknown metabolites. Metabolites were grouped into one of fifteen classes based on chemical ontology and function. Bis(monoacylglycero)phosphates were over 2-fold increase in atypical (Grade II) meningiomas versus Grade I, indicating lysosomal activation. One carbon metabolism pathway showed significant upregulation in neoplastic tissue vs. tumor involved dura, as well as neoplastic cells compared to arachnoid cells, indicating folate-dependent pathways as a potential therapeutic target. Using novel combined untargeted metabolomics, we found multiple classes of metabolites that were either enriched or suppressed in neoplastic tissue and cells compared to non-neoplastic cells. Further studies are warranted for a better understanding of possible oncogenic signaling pathways and to detect potential biomarkers useful for diagnosis and treatment.