Datura metel L., a medicinal plant in the Solanaceae family, is known for its narcotic properties and a range of physiological effects. This study investigates the diversity of 25 D. metel L. accessions using twelve morphological and seven biochemical parameters. The aim is to enhance our understanding of the plant's diversity and its potential application. Twenty-five accessions of D. metel L. were cultivated under standardized conditions. Morphological traits, including plant height, leaf length, and flower dimensions, were measured. Biochemical analysis of key compounds such as scopolamine, hyoscyamine, and atropine in the seeds and leaves was performed using High-Performance Liquid Chromatography (HPLC). This study revealed significant variability in both morphological and biochemical traits among D. metel L. accessions. Apigenin (66.28%) and atropine (44.19%) exhibited the highest coefficients of variance, while flower length (8.60%) and scopolamine (7.38%) displayed the lowest. HPLC results demonstrated notable discrepancies in biochemical levels across accessions. Cluster analysis classified the accessions into four groups, showing partial alignment with their origins. This study provides valuable insights into the physiological effects and properties of D. metel L., highlighting its morphological and biochemical diversity. These findings could contribute to the plant's enhanced agricultural and medicinal applications, emphasizing the importance of precise biochemical quantification for therapeutic advancements.