Total Tau (t-Tau), hyperphosphorylated Tau (p-Tau(181P)) and beta-amyloid(1-42) in cerebrospinal fluid (CSF) have shown to be markers of neuronal and axonal degeneration in various neurological and neurodegenerative diseases. The aim of this study was to evaluate the influence of the presence of a brain tumor and hydrocephalus on t-Tau, p-Tau(181P) and beta-amyloid(1-42) levels in CSF of pediatric patients. t-Tau, p-Tau(181P) and beta-amyloid(1-42) levels were simultaneously quantified by xMAP technology in 22 lumbar and 15 ventricular CSF samples from newly diagnosed pediatric brain tumor patients and 39 lumbar and 12 ventricular CSF samples from pediatric patients without a brain tumor. t-Tau, p-Tau(181P) and beta-amyloid(1-42) levels in both lumbar and ventricular CSF were not significantly correlated with age. t-Tau levels in lumbar CSF were elevated in brain tumor patients, being especially high in medulloblastoma patients. Lumbar CSF p-Tau(181P) levels were lower in brain tumor patients compared to normal controls. Ventricular levels of t-Tau, p-Tau(181P) and beta-amyloid(1-42) were not significantly different between the brain tumor patients and non-tumor patients, but t-Tau levels were significantly increased in patients with radiological signs of hydrocephalus. Two patients with an infected ventriculo-peritoneal drain also had high CSF t-Tau levels. In conclusion, high t-Tau levels in CSF are found in pediatric patients with a brain tumor, patients with hydrocephalus and patients with a serious CNS infection, reflecting neuronal and axonal damage. Ongoing studies should determine whether these neurodegenerative markers in CSF can be used to monitor neuronal and axonal degeneration in these patients during therapy and long-term follow up.