Mitochondrial Diseases (MDs) are a group of inherited disorders caused by defective oxidative phosphorylation. They show a wide range of clinical phenotypes and extreme genetic heterogeneity that makes the development of efficient treatments particularly difficult. Gene/mutation independent approaches acting downstream of the genetic defect are thus highly desirable. microRNAs represent a promising therapeutic tool due to their capability to simultaneously regulate different pathways. We recently demonstrated that genetic inactivation of miR-181a and b (miR-181a/b) exerts a protective action on different MDs models associated with neuronal degeneration through the enhancement of mitochondrial turnover. To test if miR-181a/b downregulation may be exploited as a therapeutic strategy in MDs we generated an Adeno Associated Viral (AAV) vector expressing a miRNA sponge able to specifically bind miR-181a/b and inhibit their activity. The miR-181a/b sponge was first tested in the Ndufs4 KO mouse, a murine model of both Leber Hereditary Optic Neuropathy and Leigh syndrome, two of the most frequent MDs affecting the eye and the brain, respectively. Interestingly mice treated with the miR-181a/b sponge show increased mitochondrial turnover and a significant amelioration of the visual function and of the locomotor activity. Moreover, our data also show an excellent safety profile of the sponge therapy in the mouse retina. Finally, we also analyze the effect of miR-181a/b sponge in OPA1-deficient SHSY5Y cells, a cellular model of Dominant Optic Atrophy, the second most common mitochondrial optic neuropathy. Our result indicates the miR-181a/b-sponge increases mitochondrial biogenesis, ameliorates mitochondrial fragmentation leading to decreased apoptosis. These findings demonstrate the therapeutic efficacy of the miR-181a/b-sponge, highlighting its efficacy as a potential new gene-independent therapeutic strategy for MDs, and unveiling the possibility to translate it in clinics.