Keratoconus (KC) is characterised by corneal stromal thinning and irregular astigmatism, resulting in diminution of vision. Underlying aetiology remains poorly understood. Recent evidence suggests dysregulation of oxidative balance and mitochondrial function in KC corneas. Therefore, this study aims to investigate the morphology of mitochondria in the corneal tissues. Twenty patients each diagnosed with KC and age-matched healthy controls were enrolled in this study. Demographic and clinical details were recorded. Corneal tissues were collected and analyzed using hematoxylin and eosin staining and transmission electron microscopy (TEM). Patients were followed up for 6 months. There was a male preponderance (75%) with an average age of 23.05 ± 6.92 years in KC patients. The clinical findings showed a history of eye atopy (65%), frequent eye rubbing (65%), and characteristic KC signs such as Munson's sign (60%) and Vogt striae (60%). The TEM analysis revealed that KC corneal epithelium exhibited numerous degraded mitochondria with dissolved cristae and heterogenous morphology, while healthy controls displayed intact mitochondrial structures. Similarly, the stroma of KC patients showed very few mitochondria with altered morphology, necrotic keratocytes, and loosely packed collagen fibrils. All the cases showed no disease progression on follow up. This is the first study providing the novel insights into the ultrastructural changes of mitochondria in epithelium and stromal layers of moderate and advanced cases of KC. This emphasises the significant mitochondrial degradation and morphological abnormalities in the corneal tissues of KC patients, indicating a pivotal role of mitochondrial dysfunction in the pathophysiology of keratoconus.