Background and Objectives: Post-surgical central nervous system (CNS) infections are severe complications associated with high morbidity and mortality. Vancomycin is a key antibiotic used in their management. However, because of the restrictive properties of the blood–brain barrier (BBB), plasma concentrations may not accurately reflect drug exposure in the brain extracellular fluid (ECF), the presumed site of infection. Cerebral microdialysis enables direct measurement of unbound drug levels in brain ECF. This study aimed to assess vancomycin penetration into brain ECF in patients with suspected or confirmed post-surgical CNS infection. Materials and Methods: Five patients with suspected or confirmed post-surgical CNS infections were enrolled. Paired brain ECF microdialysate and plasma samples (and cerebrospinal fluid (CSF) samples, when available) were collected over two consecutive days at vancomycin steady state. Vancomycin concentrations were determined using a homogeneous enzyme immunoassay and corrected for probe recovery based on in vitro calibration. Pharmacokinetic parameters, including mean concentrations and 24-h area under the concentration–time curve (AUC24), were calculated for plasma and ECF, and ECF-to-plasma ratios were derived. Results: Two subgroups could be identified: patients with negligible ECF concentrations (“low penetrators”), and those with higher ECF levels (“high penetrators”). Mean (SD) ECF-to-plasma concentration ratios were 0.07 (0.04) in “low penetrators” and 0.44 (0.10) in “high penetrators”. The corresponding AUC24 ratios were 0.06 (0.03) and 0.40 (0.03), respectively. The presence of systemic inflammatory response syndrome (SIRS) was considered the most plausible factor differentiating these two subgroups. Conclusions: Vancomycin exposure in brain ECF demonstrated substantial interpatient variability in post-surgical CNS infections, with some patients showing minimal drug penetration.