The current study was assigned to evaluate the total phenol, total flavonoid content (TPC, TFC) and antioxidant properties of extracts from the aerial parts of Scrophularia frigida (S. frigida). Extracts were also tested by preliminary phytochemical screening as well as cytotoxic activity against Artemia salina, MCF-7 (human breast carcinoma) and SW-480 (colon carcinoma) and L-929 (normal) cell lines along with antimicrobial characteristic. DPPH, MTT and Brine shrimp lethality tests and disc diffusion method were carried out to determine the biological activities of the different extracts of S. frigida. In addition, the extracts which had more potent antioxidant and antiproliferative activity were further analyzed by NMR and GC-MS. 40% methanol-water (from MeOH extract) fraction showed higher amounts of TPC, TFC and antioxidant property. Findings of the study for general toxicity effect showed that dichloromethane (DCM) and MeOH extracts had weak to moderate effects. Furthermore, DCM extract indicated the most potent anti-proliferative activity against cancer cell lines. No evidence of antibacterial activity was determined. On the other hand, analysis of the potent extract DCM in cytotoxic assay showed the presence of trans-phytol and cis-oleic acid in GCMS. Furthermore, NMR analysis of potent methanolic fractions in antioxidant tests revealed the presence of iridoids and phenolics. Generally, the results of TPC, TFC and antioxidant activity of extracts and fractions were in agreement with each other.