(1) Background: The benefit of using inhibitors of carbonic anhydrase (CA), such as acetazolamide, in the treatment of epilepsy has previously been described. (2) Methods: In this paper, the effect on CA of the most well-known antiepileptic drugs was studied in vitro and in vivo. The effects, after chronic treatment, of carbamazepine, phenytoin, valproate, primidone, clonazepam, and ethosuximide were studied in vitro on purified CA, isozyme I (CA I) and CA, and isozyme II (CA II) activity and in vivo on epileptic erythrocyte CA I and CA II activity. (3) Results: In vitro results showed that all antiepileptic drugs reduced purified CA II activity according to dose-response relationships and slightly inhibited CA I activity. In vivo results showed that the chronic administration of antiseizure drugs induced a progressive reduction in erythrocyte CA II activity in all the groups studied. This study shows that CA II inhibition can be induced both in vitro and in vivo by major antiepileptic agents as it might be one of the effective mechanisms of these anticonvulsant drugs. (4) Conclusions: The decrease in CA II activity in epileptic patients after antiseizure treatment suggests the involvement of CA II in the pathogenesis of epilepsy.