Axonal injury is believed to be a major determinant of adverse outcomes following traumatic brain injury. However, it has been difficult to assess acutely the severity of axonal injury in human traumatic brain injury patients. We hypothesized that microdialysis-based measurements of the brain extracellular fluid levels of tau and neurofilament light chain, two low molecular weight axonal proteins, could be helpful in this regard. To test this hypothesis, 100 kDa cut-off microdialysis catheters were placed in 16 patients with severe traumatic brain injury at two neurological/neurosurgical intensive care units. Tau levels in the microdialysis samples were highest early and fell over time in all patients. Initial tau levels were >3-fold higher in patients with microdialysis catheters placed in pericontusional regions than in patients in whom catheters were placed in normal-appearing right frontal lobe tissue (P = 0.005). Tau levels and neurofilament light-chain levels were positively correlated (r = 0.6, P = 0.013). Neurofilament light-chain levels were also higher in patients with pericontusional catheters (P = 0.04). Interestingly, initial tau levels were inversely correlated with initial amyloid-β levels measured in the same samples (r = -0.87, P = 0.000023). This could be due to reduced synaptic activity in areas with substantial axonal injury, as amyloid-β release is closely coupled with synaptic activity. Importantly, high initial tau levels correlated with worse clinical outcomes, as assessed using the Glasgow Outcome Scale 6 months after injury (r = -0.6, P = 0.018). Taken together, our data add support for the hypothesis that axonal injury may be related to long-term impairments following traumatic brain injury. Microdialysis-based measurement of tau levels in the brain extracellular space may be a useful way to assess the severity of axonal injury acutely in the intensive care unit. Further studies with larger numbers of patients will be required to assess the reproducibility of these findings and to determine whether this approach provides added value when combined with clinical and radiological information.