Aims: The present work investigates the implication of leaf spot disease on the antioxidant potential and commercial value of pharmaceutically important constituents of Withania somnifera, a high-valued medicinal plant. Methods and Results: Leaf spot disease was induced in W. somnifera by inoculating Alternaria alternata (Fr.) Keiss. pathogen. Total polyphenolic content and antioxidant potential showed a significant decrease during leaf spot disease. Evaluation of pharmaceutically active constituents withaferin A, withanone and withanolide A utilizing high-performance liquid chromatography showed a significant decrease in diseased samples as compared to healthy ones. Quantitative expression of major genes involved in withanolide biosynthesis also showed down-regulation in diseased samples. Alterations in the ultra-structure of chloroplasts were also analysed under transmission electron microscopy to get a better insight into the changes of withanolide biosynthesis in leaf during disease infestation. Conclusions: The present work suggests that when the pathogenic fungus invades the host plants, it evokes multiple responses, which could be studied at various levels. The knowledge gained from this work will provide appropriate rationale for controlling the bio-deterioration of the pharmaceutically active metabolites in W. somnifera and development of suitable strategies against leaf spot disease. Significance and Impact of the Study: This is the first study to investigate the effect of leaf spot disease on the human health-promoting constituents and withanolide biosynthesis in this high-valued medicinal plant.