Large quantities of spent coffee grounds (SCG) are generated the last decades, and their recycling is of research interest challenge. In the present study, SCG was tested to substitute peat (P) in substrate mixtures for the production of Brassica seedlings. Seeds of cauliflower, broccoli, and cabbage were placed in substrate mixtures containing 0-2.5-5-10% SCG. The mixture of SCG with peat affected several physicochemical characteristics of the growing media, providing also considerable amount of mineral elements for the seedling growth needs. Seed emergence was stimulated in 2.5-5% of SCG for cauliflower and at 2.5% of SCG for cabbage, while 10% of SCG decreased the percentage and increased the mean emergence time of the examined species. Plant biomass and leaf number were increased at 2.5% SCG for broccoli and cabbage but maintained at cauliflower when compared with control. The SCG at 10% decreased stomatal conductance of broccoli and cabbage (including 2.5-5% SCG in cauliflower) while chlorophyll content was increased at 10% of SCG media. The incorporation of SCG impacted the mineral content accumulated in plants with increases in nitrogen, potassium, and phosphorus and decreases in magnesium and iron content. Total phenolics and antioxidant activity (DPPH, FRAP) decreased at ≥ 5% SCG at cauliflower and cabbage or unchanged for broccoli when compared with the control. The cabbage seedlings grown in 10% SCG media subjected to stress with increases in the production of hydrogen peroxides and lipid peroxidation, and reflected changes in the antioxidant enzymatic metabolism (catalase, superoxide dismutase). The present study demonstrates that SCG (up to 5%) can be used for seed germination biostimulants and/or partially substitute the peat for Brassica seedling production.