A comprehensive investigation of the chemical composition of the aerial parts of Hypericum perforatum L. collected in three habitations in Estonia was carried out. An analysis by gas chromatography-mass spectrometry and gas chromatography-flame ionisation detection established the main components of the essential oils. The phenolic compounds both in ethanol and water extracts of the plant were analysed using liquid chromatography-mass spectrometry (LC-MS) and capillary zone electrophoresis. In addition to the earlier published polyphenols, several new phenolic acids and flavonoids, such as quercetin hexoside malonates and an A-type catechin-epicatechin trimer were identified in this Hypericum for the first time. The contents of the pharmaceutically important antidepressants hyperforin and hypericin were also estimated by LC-MS and compared with the data in the literature. The composition of the mineral elements was determined by atomic absorption spectroscopy. The results of the study demonstrate a rather high variability in the content of different substance groups in H. perforatum L. and, hence, the need for a survey of the raw material in the course of selection of raw materials for pharmaceutical preparations.