We explored the effect of unilateral intracarotid sodium amobarbital injection during the Wada test (intra-arterial amobarbital procedure, IAP) on functional connectivity in the brain assessed by synchronization analysis of the EEG. Patients suffering from pharmaco-resistant epilepsy who were selected for epilepsy surgery and underwent a preoperative IAP to determine language dominance and contralateral memory capacity were eligible. All patients had brain abnormalities (mostly tumors) or mesial temporal sclerosis. Ipsilateral intrahemispheric, contralateral intrahemispheric, and interhemispheric synchronization likelihood (SL) was calculated in seven frequency bands before and during the IAP. Forty-two patients who underwent the IAP (34 left carotid injections, 32 right carotid injections) were included. In the delta and theta bands, SL increased over the hemisphere ipsilateral to injection, while contralateral and interhemispheric SL decreased. The SL increased in the beta band. In the gamma bands, differences between patients with right-sided and left-sided lesions were observed. When a left hemisphere lesion was present, SL increased after injection, while a more unequivocal pattern of change was present in patients with right hemisphere lesions. Our results indicate that amobarbital injection has effects on functional connectivity of both the anaesthetized and non-anaesthetized hemispheres. Synchronization consistently increases in the injected hemisphere. Functional connectivity in the contralateral hemisphere decreases in the lower frequency bands, while it tends to increase in the beta and gamma bands (depending on lesion lateralization). These results indicate that functional connectivity in both the injected as well as in the contralateral hemisphere is strongly influenced by the IAP.