St. John's wort is a widely used medicinal plant. The quality of herbal drug, which is in most of the cases collected from nature, varies. Therefore, the aim of the present study was detailed chemical characterization of Hypericum perforatum subsp. perforatum samples collected in close time intervals during flowering and fruitification with the purpose to state the phenological stage characterized by maximum levels of active principles. The antioxidant potential and potential to inhibit biologically important enzymes, as well as the cytotoxicity and genotoxicity of the sample collected during the full flowering period, were evaluated. Data showed that the optimal period for the achieving of maximum level of active principles is the phenophase between floral budding and flowering stage. Significant antioxidant potential and the ability to inhibit biologically important enzymes (especially α-glucosidase) were recorded. The extract exhibited no genotoxicity in subcytotoxic concentrations, while increased cytotoxicity recorded in cotreatment with bleomycin on malignant cell lines was especially significant.