The importance of Hypericum L. species, being used in traditional medicine, in the scientific world is increasing day by day. Hypericum species are plants that have been used in the treatment of many diseases for a long time and have healing properties. In the current study, the essential oil compositions of Hypericum scabrum L. and H. ternatum Poulter collected from different localities in Turkey were determined by GC-MS/FID. In addition, their antioxidant, cytotoxic, and antimicrobial activities with their enzyme inhibitory potentials (cholinesterase, urease, tyrosinase, elastase, and collagenase) were investigated. Also, in vitro and in silico studies of the major components of the species have been carried out on the studied enzymes. It was determined that four H. scabrum samples mainly contained α-pinene (55.99%–62.80%) while three H. ternatum samples contained 2-methyloctane (9.45%–22.39%) and α-pinene (12.75%–33.08%). While H. scabrum essential oils possessed significant biological activity potential except for the antimicrobial activity, H. ternatum samples did not. All essential oil samples of H. scabrum exhibited a high cytotoxic effect (IC 50 21.67 ± 0.34 to 34.67 ± 0.45 μg/mL) against colon cancer cell line (HT-29) and indicated higher acetyl-(Inhibiton% 83.30 ± 1.90 to 93.08 ± 1.04) and butyryl-cholinesterase (Inhibiton% 80.58 ± 1.19 to 93.28 ± 1.99) inhibitory activity than the standard compound, galantamine. Furthermore, all samples of H. scabrum showed high tyrosinase (Inhibiton% 69.00 ± 1.64 to 95.25 ± 1.42) and elastase (Inhibiton% 27.58 ± 0.34 to 36.69 ± 0.18) inhibitory effects. These biological activity results indicated that H. scabrum essential oil could be used in the cosmetic and pharmaceutical industries.