Background: The diagnosis of meningitis in non-surgical hospitalized patients is often difficult and diagnostic accuracy of clinical, laboratory, and radiological characteristics is unknown. Aim: To assess diagnostic accuracy for individual clinical characteristics of patients suspected of non-surgical nosocomial central nervous system (CNS) infections. Methods: In a prospective multi-centre cohort study in the Netherlands with adults suspected of CNS infections, consecutive patients who underwent a lumbar puncture for the suspicion of a non-surgical nosocomial CNS infection were included. All episodes were categorized into five final clinical diagnosis categories, as reference standard: CNS infection, CNS inflammatory disease, systemic infection, other neurological disease, or non-systemic, non-neurological disease. Findings: Between 2012 and 2022, 114 out of 1275 (9%) patients included in the cohort had suspected non-surgical nosocomial CNS infection: 16 (14%) had a confirmed diagnosis, including four (25%) with bacterial meningitis, nine (56%) with viral CNS infections, two (13%) fungal meningitis, and one (6%) parasitic meningitis. Diagnostic accuracy of individual clinical characteristics was generally low. Elevated CSF leucocyte count had the highest sensitivity (81%; 95% confidence interval (CI): 54-96) and negative predictive value (NPV) (96%; 95% CI: 90-99). When combining the presence of abnormalities in neurological or CSF examination, sensitivity for diagnosing a CNS infection was 100% (95% CI: 79-100) and NPV 100% (95% CI: 78-100). CSF examination changed clinical management in 47% of patients. Conclusion: Diagnostic accuracy for individual clinical characteristics was low, with elevated CSF leucocyte count having the highest sensitivity and NPV (c) 2024 The Authors. Published by Elsevier Ltd on behalf of The Healthcare Infection Society. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).