Background <p>Mitochondrial DNA (mtDNA) diseases are heterogeneous and lack effective treatments. Their severity correlates with mutant mtDNA load. Mitophagy degrades dysfunctional mitochondria, contributing to a healthy mitochondrial pool. USP30, a mitochondrial deubiquitinase, limits mitophagy by removing the ubiquitin tagging mitochondria for degradation. We investigated whether inhibiting USP30 could enhance mitophagy and reduce mutant mtDNA load in a heteroplasmic mitochondrial disease.</p> Methods <p>Cybrids cells harboring mutant m.8993T &gt; G mtDNA - common cause of NARP syndrome and maternally inherited Leigh syndrome (MILS) - were treated with USP30 inhibitor MF-094 under glycolytic and oxidative phosphorylation conditions. On-target activity of MF-094 was assessed by mitochondrial ubiquitination (western-blot) and mitolysosome formation (microscopy). The mutation’s effects were investigated on cell proliferation and metabolism (respirometry and ATP levels). The impact of MF-094 on mutant mtDNA load and mtDNA copy number was quantified by PCR.</p> Results <p>Comparing with control cells (0% mutant mtDNA), cells with mutant mtDNA exhibited reduced proliferation and ATP levels under oxidative phosphorylation conditions; and reduced oxygen consumption, increased extracellular acidification, and sustained resazurin metabolism after mitochondrial inhibition under glycolytic conditions. MF-094 induced mitophagy via increased mitolysosome formation. Mechanistically, MF-094 showed on-target effects, increasing mitochondrial ubiquitination. However, chronic treatment (3–6 weeks) evoked only a small (5%) non-significant reduction in mutant mtDNA load.</p> Conclusions <p>Despite inducing mitophagy, the USP30 inhibitor MF-094 showed little potential to manage m.8993T &gt; G related diseases, as it did not significantly reduce the load of this NARP/MILS causing mtDNA mutation. These results highlight the complexity of mutant mtDNA management and the need for innovative strategies for these disorders.</p>