Allelopathy involves plants releasing allelochemicals, inhibiting nearby plants' growth and influencing their physiological and morphological characteristics. Donor plants with allelopathic capabilities dominate, impacting the survival of other plant species. The effect of Lepidium didymum stem aqueous extracts (SAE) on Lens culinaris and Melilotus alba was investigated in this study. The experiment involved extracting compounds from the stems of Lepidium didymum and applying varying concentrations (0.5, 1, 2, and 4%) of these stem extracts to test plant seeds and seedlings. Multiple parameters, including germination percentage, growth parameters, and biochemical activities, were measured and compared against the control group. Germination percentage, seedling growth, seedling length, fresh and dry biomass, chlorophyll, and carotenoid content decreased notably, especially at higher concentrations (4%), while proline content increased in a concentration-dependent manner. In 2021–2022, the experiment was carried out in the allelopathy laboratory department of Botany Aligarh Muslim University, Aligarh. Scanning electron microscopy revealed ultrastructural abnormalities in the roots of the treated plants as compared to the control. Using GCMS analysis, 32 volatile chemicals were found in the methanolic extract of the stem. The compounds found in the major amounts are benzyl nitrile (291.19%), lauric acid (7.86%), benzene acetic acid (6.44%), 11-bromoundecanoic acid (7.36%), palmitic acid (4.85%), dodecanoic acid (4.80%), menthol, trans-1,3, cis-1,4 (3.28%), 11-bromoundecanoic acid (7.36%), and linolenic acid (8.64%). These compounds may be responsible for imparting an allelopathic effect on the test species, Lens culinaris and Melilotus alba. Our study revealed that the stem of L. didymum possesses a high concentration of water-soluble allelochemicals, which are thought to have reduced test plants’ growth and can be used as potential weedicides.