Fentanyl and remifentanil are potent opioid widely used in routine anesthesia procedures. This study evaluates and compares the effects of fentanyl/remifentanil in isolated brain mitochondria bioenergetic status. Fentanyl and remifentanil in clinical concentrations does not interfere with rat brain isolated mitochondria. Do not withstand, fentanyl concentrations >4 microg/mL, induces an impairment of the respiratory chain characterized by a decrease in respiratory control ratio, state 3 and uncoupled respiration. Additionally, membrane potential collapses and ADP/O were reduced. Remifentanil follows the same profile but with effects at higher concentrations (>10 microg/mL). High concentrations of fentanyl and remifentanil interfere with mitochondrial electron chain (complexes III, IV) and on mitochondrial phosphorylation unit (complex V). Mitochondrial permeability transition pore was not induced by both fentanyl and remifentanil in tested concentrations. These data provide the first indication that fentanyl and remifentanil (microg/mL range) alters mitochondrial metabolism. Fentanyl showed a stronger inhibitory effect on mitochondrial bioenergetics.