This study was performed in order to evaluate in vitro antioxidant, anti-Trichomonas vaginalis, antimicrobial and cytotoxic activities of methanol extracts of aerial parts of Hypericum perforatum L., H. thymbrifolium Boiss. & Noë and H. thymopsis Boiss. The antioxidant activity of methanol extracts was tested with 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging and iron chelating assays. The total phenol and flavonoid contents were also determined spectrophotometrically. Cytotoxicity and apoptosis were evaluated on MCF-7 breast cancer and PC3 prostate cancer cell line. Antimicrobial activities were evaluated by the microdilution method on the bacterial strains Staphylococcus aureus (ATCC 29213), Enterococcus faecalis (ATCC 29212), Pseudomonas aeruginosa (ATCC 27853) Escherichia coli (ATCC 25922) and the fungal strain Candida albicans (ATCC 10231). All extracts demonstrated cytotoxic activity on PC3 prostat cancer cell line with a concentration dependent manner, while H. thymopsis extract was the most active against cancer cell growth. The obtained results of the present study revealed that the methanol extract of H. perforatum, H. thymbrifolium and H. thymopsis have significant antioxidant and cytotoxic activities. The results provided the basic research data for further phytochemical and biological activity guided investigations on these species for identication and isolation of potential drug active compounds.