Clinical signs and symptoms of central nervous system (CNS) infections in neonates are often nonspecific. Therefore, cerebrospinal fluid (CSF) analysis is performed to diagnose CNS infections. Data on combined microbiological results and their correlation with biochemical characteristics in CSF and blood in infants younger than 90 days are limited. This study provides an overview of microbiological test results, CSF- and hematological characteristics among infants with a clinically suspected CNS infection.This retrospective study included infants younger than 90 days, with a clinically suspected CNS infection who underwent a diagnostic lumbar puncture between January 2012 and January 2014. Data on the presence of microbiological pathogens in CSF, CSF inflammation markers (white blood cell [WBC] counts, protein levels and glucose CSF/serum ratio) and blood inflammatory responses (WBC count, C-reactive protein [CRP], neutrophil percentage) were collected by reviewing patient files.We included data from 576 infants (median age 12.5 days, interquartile range, 6-27 days) of whom 383 (66.5%) were born prematurely. In total, 16 bacterial pathogens (3.0%) and 21 viruses (5.5%) were detected in CSF. Escherichia coli was detected in 5 cases (1.0%), Enterovirus was detected in 12 cases (3.1%). Leucocytosis in CSF was associated with identification of a pathogen in CSF. Increased CRP was associated with the identification of a bacterial pathogen in CSF.Bacterial or viral pathogens were only identified in a small proportion of infants with a clinically suspected CNS infection. Leucocytosis in CSF was associated with CNS infection in infants. An increased CRP was indicative of bacterial meningitis.