The stress induced by allelochemicals present in stem aqueous extract (SAE) of Nicotiana plumbaginifolia on alterations in growth, ultrastructure on Cassia tora L., and mitotic changes on Allium cepa L. were inspected. Application of SAE at different concentrations (0.5, 1, 2, and 4%) expressively reduced the growth of C. tora in terms of seedling length and dry biomass. Moreover, the ultrastructural variations induced in the epidermis of Cassia leaf (adaxial and abaxial surface) of 15-day-old saplings were analyzed through scanning electron microscopy (SEM). The variations noticed are rupturing and shrinking of cells along epidermis; damaged margins, extensively curled leaf apex along with the appearance of puff-like structures, grooves, and thread-like structures on the leaf surface. The epidermal cells of samples exposed to treatment no longer appear smooth relative to control, besides showing necrosis as well. Upon exposure to different concentrations of extract, A. cepa root tip cells showed aberrations in chromosome arrangement and disparity in the shape of the interphase and prophase nuclei along various phases of mitotic cycle as compared to control. The mitotic index (MI) showed a concentration-dependent decline in onion root tips exposed to SAE. The aberrations appearing frequently were formation of multinucleated cells, sticky metaphase and anaphase with bridges, sticky telophase, disturbed polarity, etc. The results also show the induction of elongated cells, giant cells, and cells with membrane damage by extract treatment. To our knowledge, this is the first gas chromatography-mass spectrometry (GC-MS) analysis of the methanolic extract of N. plumbaginifolia stem. Overall, 62 compounds were reported, covering 99.61% of the entire constituents, which can be considered responsible for the allelopathic suppression of C. tora. The chief component was 4-tert-butylcalix[4]arene with the highest composition of 19.89%, followed by palmitic acid (12.25%), palmitoleic acid (8.23%), precocene 2 (7.53%), isophytyl acetate (4.01%), and betastigmasterol (3.95%).