Nonalcoholic steatohepatitis (NASH) is a disease in which fatty liver develops independently of alcohol intake and progresses to cirrhosis and liver cancer, and there are approximately 5 million affected people in Japan. Currently, no effective therapeutic agents have been found to improve the fibrosis of NASH, and a new approach to elucidate the pathogenesis of the disease is required. It has been suggested that this disease is associated with abnormal mitochondrial function. Therefore, we measured the expression of mitochondria-related factors and the production of mitochondria-derived reactive oxygen species (ROS) using liver organoids that can reproduce the pathology of NASH established in our laboratory. The results showed that the expression of the mitochondrial fission protein DRP1 and the production of ROS were increased compared to normal liver organoids. Furthermore, the mitochondrial fission inhibitor Mdivi-1 treatment suppressed the dendritic-like morphology of NASH organoids, suggesting that it may have anti-fibrotic effects.