Nevirapine (NVP) and efavirenz (EFV) are antiretroviral drugs belonging to potent class of non-nucleoside reverse transcriptase inhibitors (NNRTIs) widely used for the treatment human immunodeficiency virus (HIV) infection. It has been demonstrated that NVP and EFV are able to cross the blood–brain barrier and arrive at the central nervous system (CNS), causing important adverse effects related to their presence within this tissue. Considering that the exact mechanisms responsible for CNS toxicity associated with NVP and EFV remain unknown and that creatine kinase (CK) plays an important role in cell energy homeostasis, in the present work we evaluated CK activity in brain of mice after chronic administration of these drugs. Our results demonstrated that NVP and EFV significantly inhibited CK activity in cerebellum, hippocampus, striatum and cortex of mice. Although it is difficult to extrapolate our findings to the human condition, the inhibition of brain CK activity by NVP and EFV may be associated with neurological adverse symptoms of these drugs.