This study assessed the impact of full and late flowering stages on the polyphenols and enzyme inhibitory properties of Hypericum perforatum from Poland. Recognizing the significance of phenolic compounds in disease prevention and melatonin’s emerging protective role, we employed an UHPLC-MS/MS system to quantify 38 phenolic compounds, not typical of St. John’s wort, and to develop a new method for melatonin quantification. Afterward, the extracts were tested for their antioxidant capabilities (using phosphomolybdenum, DPPH, ABTS, FRAP, CUPRAC and ferrous chelating assays). Moreover, we investigated enzymes (acetylcholinesterase, butyrylcholinesterase and tyrosinase) involved in neurodegenerative disorders and (α-amylase and α-glucosidase) in diabetes. This study recognized the importance of phenolic compounds in disease prevention and explored the emerging protective role of melatonin, taking into account the floral ontogeny of the plant. Indeed, the full-flowering plant contained the greatest concentration of phenolic compounds (a total of 65,276.5 µg/g): hyperoside (18,726.59 µg/g), isoquercitrin (11,895.02 µg/g) and delphindin-3.5-diglucoside (10,619.51 µg/g), and showed the highest inhibitory enzyme activity. Moreover, only full-flowering St. John’s wort contained melatonin (40 ng/g). Our results offer additional perspectives on the chemical-biological characteristics of H. perforatum and scientific knowledge that testifies to the importance of considering plant growth conditions for the development of nutraceuticals.