Here we report a comprehensive and comparative study comprising mitochondrion quantification, reactive oxygen species (ROS) and respiratory efficiency, and quantitative mitochondrial proteomics of the wild-type and virus-infected strains of the chestnut blight fungus. Our data show that hypovirus infection increases the total number of mitochondria, lowers the general ROS level, but increases the mitochondrial respiratory efficiency. Quantification of mitochondrial proteomes revealed that a set of proteins functioning in energy metabolism and mitochondrial morphogenesis as well as virulence were regulated by the virus.