The Rhizomes of Nardostachys jatamansi D.C (Family Valerianceae) were collected from Haridwar, Uttarakhand. Aim: To identify the various volatile constituents of Nardostachys jatamansi D. C. Rhizomes and evaluation of antimicrobial activity. Method: The essential oil was collected by hydro distillation and analyzed by Gas Chromatography (GC) and Gas Chromatography-Mass Spectroscopy (GC- MS). Volatile oil constituents, antimicrobial potential of Nardostachys jatamansi D. C. Rhizomes have been studied using different concentrations of the volatile oil against some micro-organisms. Results: A total of 10 compounds were identified from their fragmentation pattern. The Identified compounds were Calarene (20.4%), Vardiflorene (12.3%), α- Panasinsen (9.7%), α- Santalene (4.6%), γ-Himachelene (17.1%), Jatamansone (7%), Ionol 4 (9.9%), 2,2,7,7- Tetramethyl tricyclo[6,2,1,0 (1,6)] undec-4-ene 3-one (1.7%), Epiglobulol (1.9%) and Resibufogenin (8.4%). Among them major constituents characterized was Calarene (20.4 %) and followed by γ-Himachelene (17.1%) and Vardiflorene (12.3 %). The volatile oil showed significant activity against all tested microorganisms. The maximum antibacterial activity was shown with 1%v/v of volatile oil on Staphylococcus aureus (12.8 mm) followed by Escherichia coli (12.4 mm), whereas maximum anti-fungal activity was shown by 1% v/v of volatile oil on Candida albicans (12.3 mm) followed by Aspergillus niger (11.9 mm). Conclusion: All identified constituents showed antibacterial activity and antifungal activity. Thus, the constituents should be further identified and analyzed by other spectrosopical techniques.