Essential oils are complex mixtures of biologically active substances used since along time as flavoring agents and constituents of number of commercial products. Currently, their importance is more highlighted due to their increasing demand for food,cosmetics and pharmaceutical industries. Recent scientific literature revealed theantimicrobial, antifungal, antioxidant and pharmacological potential of essential oils. In view of themultiple applications of essential oils, their characterization based on their chemicalprofiles, is of great importance.In the present study, indigenous specie of Cannabaceae family “Cannabis sativa” was screenedfor the isolation of essential oils. Essential oil components were fractioned, separated and isolated. The variations in the yield, chemical composition and biological activities of theessential oils and fractions were investigated in the present study with respect to temperature and technique of extraction.The hydro-distilled, steam distilled and supercritical fluid extracted essential oils were characterized fortheir physico-chemical and biological activities. It was tried to purify and separate the major constituents of Cannabis sativa essential oil by vacuum fractionation. GC-MS analysis of thechemical constituents of the isolated oils and fractions mainly revealed the presence of monoterpene hydrocarbons, sesquiterpene hydrocarbons, oxygenated monoterpenes, and oxygenated sesquiterpenes. The investigated essential oils and fractions exhibited good antioxidant, antimicrobial, cytotoxic, insecticidal, anti-inflammatory, antitumor, gastroprotective, nephroprotective, hepatoprotective activities as measured by different in- vivo and in-vitro assays and experimental models.