Salvadora persica L. root (Miswak) is a natural tooth cleaning tool which contains a lot of biologically active metabolites. The aim of this study is to find out the optimal method of using S. persica root as a mouth antimicrobial agent and to establish a new method for quantification of benzyl isothiocyanate (BICT) in Miswak dichloromethane extract (MDE) applying HPLC-UV. Non targeted metabolomic analysis via high performance liquid chromatography coupled with electro spray ionization tandem mass spectrometry (HPLC-PDA-ESI-MS/MS) was established for analysis of the instantly used powder (IUP) and the aqueous extract (AE). The antiviral and antibacterial activities were evaluated using MTT assay and broth micro dilution methods, respectively. HPLC-PDA-ESI-MS/MS tentatively identified 12 metabolites in IUP (glucotropaeolin, 8 flavonoid glycosides, 1 sulphated sugar derivative and 2 sugars) and 3 metabolites in the AE (2 sugars and 1 sulphated sugar derivative). IUP exhibited more potent antiviral activity against Herpes simplex virus type 1 (HSV-I) (34.79 %) than AE (25.23 %). It also demonstrated antibacterial activity against aerobic (Pseudomonas aeruginosa MIC of 6.25 mg/ml, and Bacillus sphaericus MIC 12.5 mg/ml) and anaerobic (Porphyromonas gingivalis MIC of 100 mg/ml) strains. The presence of glucotropaeolin in IUP only is suggested to be responsible for its potent antimicrobial activity in comparison to AE. A simple and validated method for quantification of BITC in MDE was established and 95 µg BITC was found to be in one gram of Miswak powder.