Hypericum (Hypericaceae) is a genus that comprises a high number of species around the world. In this study, the roots, aerial parts, flowers, fruits, and aerial parts with flowers from Hypericum scabrum were macerated separately by methanol and water and then fractionated by different solvents of, such as ethyl acetate, n-hexane, butanol, dichloromethane, aqueous residue sub-extracts, and ethnobotanical use. All the extracts, sub-extracts and essential oils of H. scabrum were investigated for the first time in detail for their antimicrobial, total phenolics, and antioxidant activities. Anatomical structures of the root, stem, leaf, upper and lower leaf surface, stamen, sepal, and petal of H. scabrum were examined. The biochemical layout of essential oils was determined by GC and GC/MS. The antioxidant activity was determined by free radical scavenging activity (by DPPH). Antimicrobial activity was applied against Candida albicans ATCC 10231, Escherichia coli ATCC 8739, Staphylococcus aureus ATCC 6538, Bacillus subtilis ATCC 19659, and C. tropicalis ATCC 750 using microdilution methods. The essentials of the aerial parts, flower, and fruit are characterized by the presence of monoterpene hydrocarbons, whereas roots oil include alkanes. The GC-FID and GC-MS analysis showed that major components of roots, aerial parts, flowers, and fruits oils were undecane (66.1%); alpha-pinene (17.5%), gamma-terpinene (17.4%), and alpha-thujene (16.9%); alpha-pinene (55.6%), alpha-thujene (10.9%), and gamma-terpinene (7.7%); alpha-pinene (85.2%), respectively. The aerial part sub-extracts indicated a greater level of total phenolics and antioxidant potential. The n-hexane sub-extracts (from aerial part, flower, and aerial part with flower) showed the best activity against B. subtilis, with 39.06 mu g/mL MIC value. The presented research work indicates that H. scabrum can be a novel promising resource of natural antioxidant and antimicrobial compounds.