Scrophularia khorassanica belongs to the Scrophulariaceae family that geographically grows wild only in Iran-Khorasan. There is insufficient information about its composition and antibacterial activity. Due to the application of S. khorassanica and related species in traditional medicine, the aim of this study was to determine antimicrobial effect of S. khorassanica aqueous and methanolic extracts on the growth of Escherichia coli and Staphylococcus aureus and also to determine the composition of the essential oil in-vitro. Extracts and the essential oil were prepared respectively by maceration and hydro-distillation methods. The composition of the essential oil examined by GC/MS and the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) determined by broth micro-dilution assay. Composition analysis identified a total of 28 various components in which the main components were 1,3-Bis (trimethylsilyl), benzene (34.93%), р-Cymene (12.55%), Palmitic acid (6.79), Thymol (6.22%), Linalool (4.59%), 2-undecanone(4.35%), Sorbitol(1.87%), Carvacrol (1.74%), γ-Terpinene (1.06%), β -Elemene (1.03%) and α-Pinene (0.3%). The MIC for methanolic extract against E. coli and S. aureus was 12.5 and 25 mg/ml, respectively. In the case of aqueous extract, it was estimated at 6.25 and 12.5 mg/ml, respectively. Based on these results, the aqueous extract had the strongest antibacterial effect. It was concluded that Scrophularia khorassanica extract can be used as an natural preservative