With the increase and spread of bacterial resistance, the importance of discovering and developing new antimicrobial compounds has reached a new level. One of the more recent antibacterial targets is the caseinolytic serine protease, which emerged as the target structure of a new class of antibiotics named acyldepsipeptides (ADEP). ADEP has a unique dual mode of action, activating the proteolytic core ClpP for non-regulated proteolysis and disrupting the natural interaction of ClpP with its associated ATPases, abolishing all natural functions of the protease. In its natural function, Clp protease maintains cellular homeostasis and plays a key role in various stress responses. In the presence of ADEP this balance is severely disturbed. Natural substrates accumulate while other crucial proteins are degraded in an ATP-independent, unregulated fashion. When bound to ADEP, ClpP wreaks havoc in the cell, overwhelming its coping mechanisms and ultimately leading to cell death. ClpP is conserved not only in prokaryotes but also in eukaryotes, where it is found in chloroplasts and mitochondria but not in the eukaryotic cytoplasm. Like its bacterial homolog, mitochondrial ClpP has important functions in eukaryotic cell homeostasis, such as protein quality control and regulation of several stress responses, including the mitochondrial unfolded protein response (UPRmt). Recent studies have shown that ClpP is upregulated and essential for proliferation and metastasis in several cancer cell types, making ClpP not only interesting as an antimicrobial target but also as a target for anti-cancer therapy. Here, we report on the effects of ADEP on mitochondrial ClpP compared to bacterial ClpP. ADEP can dysregulate mitochondrial ClpP in vitro and enter the eukaryotic cell, but its impact on the physiology of eukaryotic cells has not been studied at the molecular level. Different ADEP derivatives are compared, to determine the structure-activity relationship, and assessed side-by-side with a ClpP activator from the structurally unrelated imipridone class. We aim at increasing our understanding about the physiological role of mitochondrial ClpP and how ADEP affects its function.