Cichorium spinosum L. is a perennial wild edible plant that is usually found near the coasts of the Mediterranean basin. In the present study, we evaluated the effect of the fertilization regime on the growth and chemical profile of pot-grown C. spinosum plants, as well as the effect of extraction protocol (aqueous and hydroethanolic extracts on bioactive properties). For this purpose, plants were fertilized via a nutrient solution that differed in the amounts (mg/L) of N:P:K, e.g., 100:100:100 (C111), 200:100:100 (C211), 200:200:200 (C222), 300:100:100 (C311), 300:200:200 (C322), and 300:300:300 (C333) mg/L of N:P:K, as well as a control treatment with no fertilizer added (C0). The fertilization regime had a beneficial effect on the growth parameters of spiny chicory, while it improved its nutritional value, as indicated by the polyunsaturated (PUFA)/saturated (SFA) ratio being higher than 0.45 and the omega-6 (n-6)/omega-3 (n-3) ratio being lower than 4.0. Seven phenolic compounds were detected, including two phenolic acids and five flavonoids, while a varied composition was recorded depending on the fertilization regime and the extraction protocol. In regards to the studied bioactive parameters, antioxidant activity was significantly affected by the applied fertilizers and the extraction protocol, while there was no significant effect on the cytotoxicity, hepatotoxicity, and anti-inflammatory activity. The antimicrobial properties of C. spinosum showed varying trends depending on the bacterial strain, the fertilization regime, and the extraction protocol, whereas we recorded the extracts’ weak antifungal activity against the studied fungi. In conclusion, even though the fertilization of C. spinosum plants had beneficial effects on growth and nutritional value, a significant effect of the extraction protocol on the chemical profile and bioactivities of the edible leaves was also recorded, indicating the application of tailor-made fertilization regimes combined with the most suitable extraction method for the achievement of high-quality final products.