CXCL13 in cerebrospinal fluid has gradually become an established biomarker for Lyme neuroborreliosis (LNB), however the diagnostic performance of CXCL13 may be improved by the addition of IL-6, a non-specific infection biomarker. The aim of this study was to measure the concentrations of CXCL13 and IL-6 in cerebrospinal fluid, in the attempt to evaluate the diagnostic performance of these two biomarkers, in the differentiation between definite and possible LNB, as well as between LNB and other neuroinfections. This study used a cross-sectional design to quantify the levels of CXCL13 and IL-6 in cerebrospinal fluid (CSF) specimens from consecutive patients examined for central nervous system (CNS) infections at Lillebaelt Hospital in the Region of Southern Denmark. CXCL13 and IL-6 were measured simultaneously using the Bio-Plex 200 multiplex Cytokine Immunoassay System (Bio-Rad). Based on clinical and paraclinical findings, we grouped patients into six separate groups: definite LNB, possible LNB, Viral CNS infection, non-Borrelia Bacterial CNS infection, Other CNS disease (with pleocytosis) and Negative (without pleocytosis). A combined interpretation of four variables (leukocyte cell counts, protein concentration, CXCL13 and IL-6 concentrations in CSF) is presented using principal component cluster analysis. We included by chart review 390 patients discharged with definite LNB (n = 31), possible LNB (n = 10), confirmed Viral or non-Borrelia Bacterial CNS infection (n = 34), Other CNS disease (n = 58), and Negative (n = 257) for CXCL13 and IL-6 analysis. Principal component analysis (PCA) revealed three distinct clusters based on leukocyte cell counts, protein concentration, CXCL13 and IL-6 concentrations in CSF from 380 included patients (10 possible LNB patients excluded). The clusters clearly differentiate the groups: definite LNB, non-Borrelia Bacterial CNS infection and Negative (without pleocytosis). A receiver operating characteristic (ROC) curve comparing LNB patients (n = 31) and all non-LNB conditions with CSF pleocytosis (n = 99) indicated an optimal CXCL13 cut-off value of 50.7 pg/mL, resulting in a sensitivity and a specificity of 93.6 and 91.1%, respectively. The ROC analysis comparing patients with confirmed non-LNB CNS infection (n = 34) and all others with CSF pleocytosis (n = 97) resulted in an optimal IL-6 cut-off value of 111.5 pg/mL, yielding a sensitivity and a specificity of 78.8% and 82.5% respectively. Of the ten possible LNB patients, three cases (with CXCL13 levels above cut-off) fall within the LNB cluster, and one case is just outside, providing some laboratory support for the diagnosis of LNB. The remaining six possible LNB patients (with CXCL13 levels below the 50.7 cut-off) had little support for the diagnosis of LNB in the PCA-plot. The results of this study confirm that CXCL13 is a valuable supplement for diagnosis of LNB, and that the combination of CXCL13 and IL-6 may be used to differentiate cases of LNB from other CNS infections. Furthermore, IL-6 can be of differential diagnostic value when evaluating patients with possible LNB.