Abstract The chemical composition of methanolic extract of Hypericum pamphylicum (HPM) was analyzed by using high performance liquid chromatography–diode array detector (HPLC-DAD). The plant contained hypericin (0.00002 ± 0.000001%), hyperforin (trace), chlorogenic acid (5.58 ± 1.59%), hyperoside (0.46 ± 0.05%), isoquercitrin (0.134 ± 0.02%) and quercetin (0.087 ± 0.008%). The antioxidant activity of HPM was evaluated by using several antioxidant tests. HPM exhibits inhibitory capacity against phosphatidylcholine liposome peroxidation, induced with iron and ascorbic acid, scavenge DPPH and superoxide radicals and act as reductants. The cytotoxic activity of HPM was also determined by using MTT cell viability assay on HeLa and NRK-52E cell lines. HPM demonstrated no cytotoxicity against HeLa and NRK-52E cell lines with IC50 values of 1.89 mg/ml and 1.05 mg/ml, respectively. The different crude extracts of H. pamphylicum were analyzed using a microdilution assay for antimicrobial activity against several microorganisms. The most active extract was HPM which showed activity against Staphylococcus aureus. It is observed that nearly all extracts showed activity against Staphylococcus epidermidis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella thypi, Shigella flexneri, Proteus mirabilis and Candida albicans.