Background Magnetic resonance spectroscopy (MRS) is usually added to conventional magnetic resonance imaging (MRI) to refine the diagnosis of different brain lesions. Stereotactic brain biopsy is a well-established method to obtain tissues for histopathologic examination. The purpose of the study is to compare the diagnostic yields of MRS and stereotactic biopsy in the characterization of brain lesions. Material and Methods A prospective study conducted on 27 consecutive patients presenting with multifocal, diffuse, as well as deeply seated intra-axial brain lesions. All patients had both brain MRI and MRS prior to stereotactic biopsy. Histopathologic examinations of the obtained tissue specimens, using appropriate stains including immunostains, were performed. Results MRS diagnosed neoplastic brain lesions in 15 cases (56%) and nonneoplastic brain lesions in 12 (44%). Correlation between the preoperative diagnosis by MRS and the histopathologic diagnosis following stereotactic biopsy of either a neoplastic or nonneoplastic lesion revealed matching in 25 of 27 cases (sensitivity 88%; specificity 100%). Within the group of cases (n = 15) diagnosed preoperatively by MRS as neoplastic, 12 patients were diagnosed with brain gliomas of different grades. The MRS grading of gliomas exactly matched the histopathologic grading following stereotactic biopsy in 10 of the 12 cases (sensitivity 89%; specificity 67%). Conclusions MRS is a useful addition to the management armamentarium, providing molecular information that assists in the characterization of various brain lesions. Multivoxel MRS may increase the diagnostic yield of stereotactic biopsy by guidance to target the higher choline and lower N-acetylaspartate areas, expected to have greater tumor activity.