Cymbopogon flexuosus is a medicinal plant with various biological activities. Although its essential oil (EO) is widely used, the effect of C. flexuosus EO on mitochondrial bioenergetics has not been completely elucidated. Here, we report the effect of C. flexuosus EO on electron transport through the mitochondrial respiratory chain using isolated mitochondria from liver rats. The EO decreases the oxygen consumption rate up to −20% at 10 ppm, and −17% at 25 ppm using glutamate and succinate, respectively. The C. flexuosus EO inhibits the NADH oxidase (34%), succinate oxidase (53%), succinate dehydrogenase (44%), and cytochrome c oxidase (69%) activities at 10 ppm, which indicates a robust inhibitory effect on the electron transport chain. Our results show the inhibitory effect of C. flexuosus EO on electron transport through complex II and IV and suggest that this EO could be used as a potential drug to treat mitochondrial diseases, including cancer.