Mitochondria play a vital role in fundamental cellular processes, serving as key regulators of energy metabolism, apoptosis, oxidative stress, calcium homeostasis. Mitochondrial dysfunction is widely regarded as a common pathogenic pathway in the development of widespread chronic diseases, such as metabolic disorders, cardiovascular disease, neurodegeneration, and malignancies. Modern research examines mitochondrial dynamics, mitophagy, mitochondrial biogenesis, mtDNA damage, and the role of reactive oxygen species not only for in-depth understanding of disease pathogenesis but also for identifying diagnostic markers and therapeutic targets. Determining mitochondrial dysfunction is a significant challenge and should involve a comprehensive approach with reliable assessment methods that take into account the dynamic state, number, and localization of mitochondria. The review summarizes the results of the studies exploring the pathogenetic role of mitochondrial dysfunction in the development of widespread chronic diseases and current methods of its evaluation for the integration of mitochondrial dysfunction biomarkers into modern diagnostic strategies and development of mitochondria-target treatment approaches.