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Essential oils are secondary metabolites of various parts of plants. Essential oil extracted from plants have unique therapeutic benefits. These are used in various forms such as lotions ,ointment, lubricants, perfumes, fragrant bath perfumed water, massage and incense. The antimicrobial action of essential oil is one of the most extensively studied aspects of plant medicine. Dried tuberous roots of Cyperus rotundus is known as source of aromatics, used in perfumes and agarbatties. The reported constituents of volatile oil in Cyperus rotundu tubers vary with the source so in this study, the composition of essential oil obtained from Cyperus rotundus Linn. growing in Dehradun (Uttarakhand), India, a new location is compared with the previous study along with its antimicrobial activity.