The Mediterranean basin is abundant in wild edible species with numerous health beneficial effects due to the presence of various bioactive phytochemicals. In the present work, the effect of nitrogen fertilization rates (0 ppm, (N0), 200 ppm (N1), 400 ppm (N2), and 600 ppm (N3) of total N) and growth substrate composition (soil or peat/perlite (2/1; v/v)) on the chemical composition and bioactive properties of Centaurea raphanina ssp. mixta plants was evaluated. The results of the study showed that both the tested factors affected nutritional value of the edible leaves, with the soil × N1 treatment being the most beneficial for fat, protein, and carbohydrate content and energetic value. On the other hand, the peat/perlite-grown plants that received 200 ppm of N had the highest content in α-, γ-, and total tocopherols, while the control treatment of soil-grown plants was the richest in individual and total sugars. Oxalic, citric, and total organic acids were the highest in the N2 × soil treatment, while malic acid was the highest in control treatment of the same substrate. The main fatty acids were palmitic, α-linolenic, and linoleic acids, with the highest contents being observed in the N0 × soil, N3 × soil, and N3 × peat/perlite treatments, respectively. The major phenolic compounds were pinocembrim neohesperidoside and pinocembrim acetyl neohesperidoside isomer II, with the highest content being observed in the N1 × soil treatment. The highest antihemolytic activity was observed in the N3 × peat/perlite treatment, while the most effective treatments against lipid peroxidation were N0 (in both soil and peat/perlite combinations) and N1 × peat/perlite. Lastly, all the tested extracts (except for N1 × soil) showed promising cytotoxic effects against HeLa (cervical carcinoma), HepG2 (hepatocellular carcinoma), MCF-7 (breast carcinoma), and NCI-H460 (non-small-cell lung cancer), while all the tested extracts exhibited better antifungal activities (lower minimal inhibition concentration (MIC) values) against Trichoderma viride than the positive controls. Overall, the present results suggest that the application of cost-effective practices such as the nitrogen application and the selection of growth substrate may regulate the chemical composition and the bioactive properties of C. raphanina ssp. mixta species and increase its added value under commercial cultivation conditions.