Following brain injury increased glutamate release is linked to sustained neuronal activation resulting in excitotoxic tissue damage. Isoflurane anesthesia has been shown to decrease electroencephalographic (EEG) activity and extracellular (e.c.) glutamate, possibly attenuating excitotoxic tissue damage. However, based on clinical experience EEG activity can fluctuate despite stable isoflurane concentrations. Therefore, the aims of this study were to investigate the impact of neuronal activity assessed by quantitative EEG on e.c. glutamate during isoflurane anesthesia following controlled cortical impact injury (CCII). In 10 rats balanced anesthesia using isoflurane was induced at 4 hours after CCII. Under steady-state conditions maintaining isoflurane at 1.8 vol%, EEG was recorded for 3 hours. During this period, e.c. glutamate was sampled in the pericontusional cortex by microdialysis. Despite maintaining isoflurane at 1.8 vol%, neuronal activity expressed as low frequency EEG power showed marked fluctuations. Spontaneous increases in neuronal activity coincided with elevated e.c. glutamate levels and vice versa. Overall, EEG power correlated significantly with pathologically elevated e.c. glutamate levels (n = 58; R2 = 0.54; p < 0.05). Despite unchanged isoflurance concentrations fluctuations in neuronal activity were reflected by altered EEG power and e.c. glutamate concentrations. Therefore, neuronal activity needs to be considered for the interpretation of e.c. glutamate levels.