ObjectiveTo characterize the microbiota of the cerebrospinal fluid (CSF) from children with hydrocephalus at the time of initial surgical intervention.Study designCSF was obtained at initial surgical intervention. One aliquot was stored in skim milk-tryptone-glucose-glycerol (STGG) medium and the second was unprocessed; both were then stored at –70°C. Bacterial growth for CSF samples stored in STGG were subsequently characterized using aerobic and anaerobic culture on blood agar and MALDI-TOF sequencing. All unprocessed CSF samples underwent 16S quantitative polymerase chain reaction (qPCR) sequencing, and a subset underwent standard clinical microbiological culture. CSF with culture growth (either after storage in STGG or standard clinical) were further analyzed using whole-genome amplification sequencing (WGAS).Results11/66 (17%) samples stored in STGG and 1/36 (3%) that underwent standard clinical microbiological culture demonstrated bacterial growth. Of the organisms present, 8 were common skin flora and 4 were potential pathogens; only 1 was also qPCR positive. WGAS findings and STGG culture findings were concordant for only 1 sample, identifyingStaphylococcus epidermidis. No significant difference in time to second surgical intervention was observed between the STGG culture-positive and negative groups.Conclusion(s)Using high sensitivity methods, we detected the presence of bacteria in a subset of CSF samples at the time of first surgery. Therefore, the true presence of bacteria in CSF of children with hydrocephalus cannot be ruled out, though our findings may suggest these bacteria are contaminants or false positives of the detection methods. Regardless of origin, the detection of microbiota in the CSF of these children may not have any clinical significance.