In this study, the chemical characterization and bioactive properties of S. minor cultivated under different fertilization rates (control, half rate and full rate) were evaluated. Twenty-two phenolic compounds were identified, including five phenolic acids, seven flavonoids and ten tannins. Hydrolysable tannins were prevalent, namely Sanguiin H-10, especially in leaves without fertilization (control). Roots of full-rate fertilizer (660 Kg/ha) presented the highest flavonoid content, mainly catechin and its isomers, whereas half-rate fertilizer (330 Kg/ha), presented the highest content of total phenolic compounds, due to the higher amount of ellagitannins (lambertianin C: 84 ± 1 mg/g of dry extract). Antimicrobial activities were also promising, especially against Salmonella typhimurium (MBC = 0.44 mg/mL). Moreover, root samples revealed activity against all tested cell lines regardless of fertilization rate, whereas leaves were effective only against HeLa cell line. In conclusion, S. minor could be a source of natural bioactive compounds, while fertilization could increase phenolic compounds content.