Background As biostimulants have become a strategic tool for sustainable crop production in the face of changing environmental conditions, there is a continual demand for the development of innovative and more effective formulations. A novel approach that utilizes plasma treatment to enhance the extraction process in biostimulant production has recently emerged. This study investigated the plant-based biostimulant (prepared from horsetail, dog rose, and soapwort) and the potential of using gliding arc cold plasma (GA) and low-pressure microwave (MW) discharges to improve its efficacy. The experiment evaluated the effects of untreated versus plasma-treated biostimulants on soybean plant growth and vitality while elucidating their mechanisms. Results The results revealed that biostimulant application had a positive effect (p < 0.05) on different microbial groups in the soil. The treated plants presented greater root length and biomass (p < 0.05) as well as increased shoot height (p < 0.05). Generally, treatment with biostimulants increased lignification, as reflected by higher acid detergent fibre (ADF) and acid detergent lignin (ADL) fractions, as well as elevated peroxide levels, total isoflavone content, and antioxidant potential of plants measured by DPPH and ABTS assays. All biostimulants strongly upregulated the pathogenesis-related protein genes CHIA1 (encoding chitinase A1) and GLU (encoding β-1,3-glucanase) in the leaves of soybean plants. Elevated expression of selected genes related to jasmonic acid (JA) biosynthesis, mitogen-activated protein kinase (MAPK) signalling, cellular detoxification, and redox homeostasis was also observed. Conclusions Overall, all the tested biostimulant variants improved the growth parameters of the soybean plants. Biostimulant application triggered complex defence responses involving changes in gene expression, antioxidant potential, secondary metabolite levels, and cell wall composition. The formulation generated by GA outperformed the other tested variants (MW discharge and untreated biostimulants), highlighting the potential of this technology to enhance biostimulant efficacy.