The differential diagnosis of acute community-acquired meningitis is of paramount importance in both therapeutic and healthcare-related economic terms. Despite the routinely used markers, novel, easily calculated, and rapidly available biomarkers are needed particularly in resource-poor settings. A promising, exponentially studied inflammatory marker is the neutrophil-to-lymphocyte ratio (NLR), albeit not assessed in meningitis. The aim of this study was to investigate the utility of the NLR in the differential diagnosis of acute meningitis. Data on cerebrospinal fluid (CSF) and blood leukocyte parameters from more than 4,000 patients diagnosed with either bacterial or viral meningitis in Greece during the period 2006-2013 were retrospectively examined. The diagnostic accuracy of the NLR and neutrophil counts in CSF and blood were evaluated by receiver operating characteristic curves. The discrimination ability of both the NLR and neutrophil counts was significantly higher in CSF than in blood. The optimal cutoff values of the NLR and neutrophil counts were 2 in CSF vs 8 in blood, and 287 cells in CSF vs 12,100 cells in blood, respectively. For these values, sensitivity, negative predictive value, and odds ratio were statistically significantly higher in CSF than blood for both markers. Logistic regression analysis showed that the CSF NLR carries independent and additive information to neutrophil counts in the differential diagnosis of acute meningitis. This study is the first one to assess NLR in acute meningitis, providing promising results for its differential diagnosis.