Datura Stramonium is a well-known and important medicinal plant that is widely used in various medical systems to treat conditions such as asthma, diabetes, and inflammatory diseases. The aim of this study was to prepare extracts of D. stramonium seeds in different solvent polarities for assessing phytochemical potential, in vitro biological activities, and molecular docking studies. Phytochemical screening was conducted to determine the phytochemical composition, while GC-MS analysis was used to identify secondary metabolites of D. Stramonium. The seed extracts were molecularly docked to evaluate bioactive and antioxidant activity. The minimum inhibitory concentration (MIC) of the extracts against bacteria such as S. aureus, E. coli, Bacillus subtilis, and Proteus mirabilis was determined. Antifungal activity was also tested against fungi including Aspergillus flavus, Fusarium oxysporum, Aspergillus niger, and Candida albicans. The results of phytochemical screening indicated that the primary plant constituents in all extracts of different polarities are alkaloids, flavonoids, tannins, mucilage, sterols, heterosides, triterpenoids, and cardiac glycosides. Most molecules in the hexane, ether, and chloroform extracts consist of fatty acids, sterols, glycosides, triterpenoids, alkaloids, and phenolic compounds, as revealed by GC-MS analysis. Conversely, extracts from polar solvents like methanol, ethanol, and water are abundant in alkaloids. In vitro antibacterial and antifungal activities demonstrated that ether, methanol, and ethanol extracts were more effective than inhibiting the four bacterial strains compared to the antibiotics Oxacillin and Cefuroxime. The ether and methanol extracts exhibited better zones of inhibition and significant MIC values against A. niger and C. albicans compared to the control and fluconazole. The free radical inhibition (DPPH) (24.92 ± 5.31 μg/mL) for the ethanol extract indicates Datura's antioxidant capacity. It is believed that the main phytochemicals are responsible for the enhanced antioxidant activity observed in other studies. The docking study revealed that the bioactive compounds linoleic acid and atropine formed better hydrogen bonding interactions with proteins than pi-alkyl and alkyl bonds.