Objective The genus Scrophularia is one of the largest genera of the Scrophulariaceae family that about 60 species of this genus grow in Iran. Scrophularia frigida and Scrophularia subaphylla are two of endemic species growing naturally in Iran. Several members of this genus are known to possess medicinal uses as anticancer, anti-inflammatory, antibacterial, cardiovascular, diuretic and protozoacidal in traditional medicine. Scope Regarding the importance of reported biological effects of isolated compounds from this genus, it sems to be rational to start phytochemical and biological studies on the species growing naturally in the country. Experimental The dried and ground aerial parts of these two species were extracted successively using n-hexane, dichlomethane ( DCM) and methanol (MeOH) by soxhlet extraction. Dried MeOH extracts were subjected to C18 Sep-Pak fractionation using a step gradient of MeOH-H2O (19, 28, 46, 64, 82, 100). Further purification of the fractions by preparative reversed-phase HPLC yielded 18 compounds. Structure elucidation for all of these compounds were accomplished by using 1HNMR and 13CNMR. Furthermore, the essential oils were obtained by hydrodistillation in a Clevenger extractor, as well. The composition of essential oils were determined by coupled GC/MS. Additionally, antioxidant, general toxicity and anti-malarial activity assays using DPPH, Brine Shrimp Lethality and Cell Free Beta Hematine methods, were performed on extracts. Results Phytochemical study on S. frigida yielded phenyl ethanoid derivatives, flavonoid, acylated and non acylated iridoid glycosides. Furthermore, phytochemical analysis of S. subaphylla yielded acylated and non acylated iridoid glycosides and phenylethanoid derivatives. GC/MS analysis of the essential oils of S. frigida and S. subaphylla led to identification of 18 and 36 compounds, respectively. Antioxidant and general toxicity assays revealed good activities of MeOH extracts in both species. Moreover, among the extracts, DCM extract illustrated good anti-malarial activity in both species. Conclusion Presence of glycosylated iridoids seems to be an important feature in both species which they may play dominant role, compared to other classes of secondary methabolites. Both species contain some phytochemicals responsible for observed biological activities.