The aim of this retrospective study was to review the diagnostic accuracy of real-time polymerase chain reaction (PCR) testing of cerebrospinal fluid (CSF) samples for Streptococcus pneumoniae DNA in comparison with traditional bacterial culture. The hypothesis was that PCR is more sensitive than culture and would detect more cases of pneumococcal meningitis, particularly in children treated with antimicrobials before CSF sampling occurred. Patients younger than 16 years of age who had a CSF sample tested for S. pneumoniae DNA by PCR between 2004 and 2015 were included. A total of 2025 samples were included, and the PCR had a sensitivity of 100% and specificity of 98% for the detection of S. pneumoniae DNA in comparison with culture. Of the 28 culture negative/PCR positive cases, 25 (89%) were probable meningitis cases and only 3 (11%) were suspected false positive results. Nineteen (76%) of the 25 probable cases required ICU admission, and 3 died (12%). Six different serotypes were found in the culture positive patients (18C, 6B, 14, 22F, 7F and 33F). This study demonstrates that PCR testing of CSF samples for S. pneumoniae is sensitive and specific when compared with culture. PCR is particularly useful in detecting those cases where culture is negative, perhaps relating to pre-CSF sampling administration of antimicrobials.