OBJECTIVE: A substantial part of central nervous system (CNS) disorders remains unexplained, despite various new and minimally invasive diagnostic techniques. Within this rapidly developing diagnostic field, the precise role of brain biopsy is unknown. We aimed to study the clinical impact and safety of brain biopsies in unexplained CNS disorders. METHODS: In this retrospective cohort study, we included all adult patients who were referred for a diagnostic work-up to our academic center with neuro-inflammatory, neuro-oncological, and neuro-infectious expertise and underwent a brain biopsy between January 2010 and December 2023. Typical cases of CNS neoplasms and infections were not analyzed. Brain biopsies were evaluated with respect to diagnostic and therapeutic impact and complication risk. RESULTS: Brain biopsy was performed in 587 patients. Ninety-four patients with a CNS disorder of unknown cause, with 107 biopsies, were analyzed (44% female, median age 58 years). Postoperative diagnoses included brain tumors/lymphomas (37/94, 39%), inflammatory disorders (11/94, 12%), infections (8/94, 9%), autoimmune encephalitis (8/94, 9%), and primary angiitis of the CNS (4/94, 4%). Diagnostic yield of brain biopsy was 62%, increasing up to 72% after repeat biopsies, as 10 additional patients were diagnosed with a brain tumor. In 77% of patients, brain biopsy changed the treatment strategy. Symptomatic intracranial hemorrhage occurred in 4 of 107 brain biopsies (4%). INTERPRETATION: In a selected population of patients with unexplained CNS disorders, clinical impact of brain biopsies is high, while being relatively safe. A multidisciplinary team approach is fundamental in establishing optimal indication for brain biopsy and subsequent treatment decisions.