Background: Applications in clinical medicine can benefit from fusion of spectroscopy data with anatomical imagery. For example, new 3-dimensional (3D) spectroscopy techniques allow for improved correlation of metabolite profiles with specific regions of interest in anatomical tumor images, which can be useful in characterizing and treating heterogeneous tumors that appear structurally homogeneous. Objectives: We sought to develop a clinical workflow and uniquely capable custom software tool to integrate advanced 3-tesla 3D proton magnetic resonance spectroscopic imaging (1H-MRSI) into industry standard image-guided neuronavigation systems, especially for use in brain tumor surgery. Methods:1H-MRSI spectra from preoperative scanning on 15 patients with recurrent or newly diagnosed meningiomas were processed and analyzed, and specific voxels were selected based on their chemical contents. 3D neuronavigation overlays were then generated and applied to anatomical image data in the operating room. The proposed 3D methods fully account for scanner calibration and comprise tools that we have now made publicly available. Results: The new methods were quantitatively validated through a phantom study and applied successfully to mitigate biopsy uncertainty in a clinical study of meningiomas. Conclusions: The proposed methods improve upon the current state of the art in neuronavigation through the use of detailed 3D 1H-MRSI data. Specifically, 3D MRSI-based overlays provide comprehensive, quantitative visual cues and location information during neurosurgery, enabling a progressive new form of online spectroscopy-guided neuronavigation.