Cells can adjust their mitochondrial morphology by altering the balance between mitochondrial fission and fusion to adapt to stressful conditions. The connection between a chemical perturbation, changes in mitochondrial function, and altered mitochondrial morphology is not well understood. Here, we made use of high-throughput high-content confocal microscopy to assess the effects of distinct classes of oxidative phosphorylation (OXPHOS) complex inhibitors on mitochondrial parameters in a concentration and time resolved manner. Mitochondrial morphology phenotypes were clustered based on machine learning algorithms and mitochondrial integrity patterns were mapped. In parallel, changes in mitochondrial membrane potential (MMP), mitochondrial and cellular ATP levels, and viability were microscopically assessed. We found that inhibition of MMP, mitochondrial ATP production, and oxygen consumption rate (OCR) using sublethal concentrations of complex I and III inhibitors did not trigger mitochondrial fragmentation. Instead, complex V inhibitors that suppressed ATP and OCR but increased MMP provoked a more fragmented mitochondrial morphology. In agreement, complex V but not complex I or III inhibitors triggered proteolytic cleavage of the mitochondrial fusion protein, OPA1. The relation between increased MMP and fragmentation did not extend beyond OXPHOS complex inhibitors: increasing MMP by blocking the mPTP pore did not lead to OPA1 cleavage or mitochondrial fragmentation and the OXPHOS uncoupler FCCP was associated with OPA1 cleavage and MMP reduction. Altogether, our findings connect vital mitochondrial functions and phenotypes in a high-throughput high-content confocal microscopy approach that help understanding of chemical-induced toxicity caused by OXPHOS complex perturbing chemicals.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10565-022-09712-6.