Abstract: Background and Objective: Mentha longifolia is an extremely flavorful and aromatic herb of the mint family native to many parts of Europe, Asia and Northern and Southern Africa also known as horse mint. Extracts of M. longifolia have been used for centuries as a food flavoring and a medicine. The present study was aimed to reinvestigate the essential oil of Mentha longifolia (L.) Huds. for its chemical composition and concomitantly anti-inflammatory, analgesic, antipyretic, antibacterial and fungicidal activity. Materials and Methods: The essential oil was extracted using hydrodistillation method and analyzed by GC-MS. Anti-inflammatory, anti-nociceptive and antipyretic activities of essential oil were experimentally determined using mice model. Antibacterial activity and antifungal activity were determined by using disc diffusion and agar dilution method, respectively. Results were analyzed using one-way ANOVA followed by Dunnett’s multiple comparison test. Results: The major constituents identified in the essential oil of Chamoli collection were piperitenone oxide (60.44%), 1,8-Cineole (6.16%), piperitenone (3.46%), dl-limonene (2.72%) and piperitone (1.69%). The Joshimath collection showed the presence of piperitenone oxide (36.89%), cis-piperitone oxide (17.89%), camphor (3.11%), isophorone (2.52%), thymol (2.41%) and coumarin (1.41%) and the Badrinath collection contains cis-piperitone oxide (42.74%), piperitenone oxide (33.48%), 4-Hydroxypiperitone (3.26%), thymol (2.52%), 2-Isobutylcyclopent-3-en-1-one (1.57%) and piperitenone (1.02%). The other constituents were present in minor quantities (less than 1.0%) in all three collections. The essential oil of Mentha longifolia show insignificant anti-inflammatory and analgesic activity but shows significant antipyretic, antibacterial and antifungal activity. Conclusion: The study indicated the wide chemical diversity in the essential oil of Mentha longifolia (L.) Huds collected from different geographical regions. The variation in chemical constituents may also effect the biological activities of the samples collected from different locations.