Volatile oil composition of hydro-distilled (HD) and supercritical carbon dioxide (SC-CO2) essential oil of freshly collected aerial parts of Heracleum thomsonii (Umbeliferae) from the western Himalayas was studied by GC-FID and GC-MS. Results revealed qualitative and quantitative dissimilarity in the composition of hydro-distilled and SC-CO2 extracted oils. Nineteen constituents, which accounted for 89.32% of total constituents in HD oil, represented by limonene (4.31%), (Z)-beta -ocimene (3.69%), terpinolene (22.24%), neryl acetate (36.19%), nerol (9.51%) and p-cymene-8-ol (2.61%) were identified. In SC-CO2 extracted oil, 24 constituents representing 89.95% of total constituents were identified. Terpinolene (5.08%), germacrene D (2.17%), neryl acetate (51.62%), nerol (9.78%), geranyl acetate (2.06%), alpha-bisabolol (2.48%) and 1-nonadecanol (4.96%) were the dominating constituents. In vitro antimicrobial activity of hydro-distilled oil was conducted against microrobial strains including two Gram-positive (Staphylococcus aureus and Bacillus subtilis) and five Gram-negative (Burkholderia cepacia, Escherichia coli, Enterobacter cloacae, Klebseilla pneumoniae and Pseudomonas aeruginosa) bacteria as well as seven fungi (Candida albicans, Issatchenkia orientalis, Aspergillus flavus, Aspergillus niger, Aspergillus parasiticus, Aspergillus sydowii and Trichophyton rubrum) using broth microdilution method. The results of bioassay showed that the oil exhibited moderate to high antimicrobial activity against fungi C. albicans (MIC 625 mu gml(-1)), A. parasiticus (MIC 312.5 mu gml(-1)), A. sydowii (MIC 312.5 mu gml(-1)), T. rubrum (MIC 625 mu gml(-1)), Gram-positive bacteria B. subtilis (MIC 625 mu gml(-1)) and Gram-negative bacteria P. aeruginosa (MIC 312.5 mu gml(-1)).