Antibiotics are the first line of treatment against infections and have contributed immensely to reduce the morbidity and mortality rates. Recently, extensive use of antibiotics has led to alterations of the gut microbiome, predisposition to various diseases and most importantly, increase in the emergence of antibiotic-resistant bacteria, which poses a major threat to global public health. Another major issue faced worldwide due to unregulated use of antibiotics in children as well as in adults is the influence of metabolism and body weight homeostasis, leading to obesity. Apart from the involvement of biosocial causes influencing diet, physical activity, and antibiotic use, pathogenesis of obesity is linked to interconnected functional alterations in cells, tissues and organs due to biochemical, epigenetic and genetic factors. Mitochondrial dysfunction is one such factor, which is becoming the primary focus of various aspects of research on multifactorial complex diseases and is providing new perspectives on etiology, biomarker-based diagnosis, and drug sensitivity. Through this review, we have made an attempt to present the interplay between use of antibiotics, obesity, and associated mitochondrial dysfunction. This may provide insights into the molecular basis, genetic predisposition and environmental triggers, which in turn may have potential clinical applications in the management of antibiotic use.