Hypericum empetrifolium Willd is a member of the Hypericaceae family, mainly known in southern Greece, and western Turkey. Being a centuries-old medicinal plant, the aerial parts of the plant have been used for the treatment of herpes, kidney stones, gastric ulcer, and also for their anti-helminthic, and diuretic purposes traditionally.The current study aimed to investigate the phytochemical profiles of the essential oil, and two ethanol extracts prepared from the aerial parts (H. empetrifolium aerial parts extract - HEA), and roots of the plant (H. empetrifolium roots extract - HER), and to provide data on antioxidant, anticholinesterase, antityrosinase, antiurease, cytotoxic, and antimicrobial activities of the extracts.In this study, volatile and phenolic compounds of the HEA and HER were analyzed by GC-MS and LC-MS/MS, respectively. Antioxidant potential of the extracts was clarified by using DPPH radical scavenging assay, ABTS cation radical assay, and the CUPRAC assay. Acetylcholinesterase, butyrylcholinesterase, urease, and tyrosinase inhibitory activity assays were used to determine enzyme inhibition capacity of the extracts. Cytotoxic activity of the extracts was established by using XTT assay. The antimicrobial activity of the extracts was determined by the microbroth dilution technique.The major compounds of the essential oil were revealed as alloaromodendrene (24.7%), α-pinene (14.7%), β-pinene (10.7%), and α-terpineol (7.7%) by the GC-MS analysis. According to the LC-MS/MS analysis results, quinic acid was the most abundant constituent in both extracts with 20612.42 ± 169.02 μg/g extract in HEA extract, and with 2254.34 ± 18.49 μg/g extract in HER extract, respectively. The HEA extract was also found to be rich in terms of chlorogenic acid (5583.14 ± 38.52 μg/g extract), isoquercitrin (3076.77 ± 40.92 μg/g extract), and malic acid (2822.90 ± 31.90 μg/g extract). HEA extract exhibited a significant antioxidant effect with an IC50 value of 11.98 ± 0.22 μg/mL according to the DPPH radical scavenging assay. Similar results were obtained with the ABTS cation radical assay, and the CUPRAC assay. HER extract showed a strong butyrylcholinesterase inhibitory effect with 88.69 ± 0.62% at 200 μg/mL concentration. Both extracts were considered to have moderate anti-tyrosinase activity compared to the standard at 200 μg/mL. The best antimicrobial activity was obtained for HEA against C. parapsilosis with an MIC value of 4.88 μg/mL. HEA also exhibited antifungal activity against C. tropicalis with 19.53 μg/mL. Only HER exhibited low cytotoxicity on A498 renal cell lines with 60.6% cell viability.Unquestionably, H. empetrifolium Willd has ethnopharmacological importance according to these results, and further investigations are required to evaluate other therapeutic properties of the plant.