Mitochondria are important for the viral life cycle, mainly by providing the energy required for viral replication and assembly. A highly complex interaction with mitochondria is exerted by rubella virus (RV), which includes an increase in the mitochondrial membrane potential as a general marker for mitochondrial activity. We aimed in this study to provide a more comprehensive picture of the activity of mitochondrial respiratory chain complexes I to IV. Their activities were compared among three different cell lines. A strong and significant increase in the activity of mitochondrial respiratory enzyme succinate:ubiquinone oxidoreductase (complex II) and a moderate increase of ubiquinol:cytochrome c oxidoreductase (complex III) were detected in all cell lines. In contrast, the activity of mitochondrial respiratory enzyme cytochrome c oxidase (complex IV) was significantly decreased. The effects on mitochondrial functions appear to be RV specific, as they were absent in control infections with measles virus. Additionally, these alterations of the respiratory chain activity were not associated with an elevated transcription of oxidative stress proteins, and reactive oxygen species (ROS) were induced only marginally. Moreover, protein and/or mRNA levels of markers for mitochondrial biogenesis and structure were elevated, such as nuclear respiratory factors (NRFs) and mitofusin 2 (Mfn2). Together, these results establish a novel view on the regulation of mitochondrial functions by viruses.