Eucalyptus globules is an widely distributed in tropical and subtropical regions. It has been widely used as folk medicine, and folk cosmetic owing to its antioxidant values. Despite its importance, phytochemical and pharmacological studies remain infancy. This study was aimed at extraction of essential oil by steam-distillation and evaluation of bioactive components, antioxidant, antimicrobial, anti-inflammatory activities along with analysis by UV-VIS, FT-IR and Fluorescent techniques. Fast protein liquid chromatography (FPLC) was used to confirm the presence of polyphenols. Different antioxidant activities like DPPH., ABTS.+, .OH, superoxide, nitric oxide and reducing power of the essential oil. Essential oil was analyzed by UV-VIS, FT-IR and Fluorescent techniques. In vitro antimicrobial activity was also monitored. FT-IR fingerprint qualitative analysis was performed using commercial standards. Considerable amount of flavonoids were detected in essential oil. Oil exhibited considerable scavenging activities of ABTS.+, .OH, superoxide, nitric oxide and reducing power. UV-VIS, FT-IR analysis revealed the presence of polyphenolics in essential oil. Fluorescent spectroscopy revealed the presence of fluorophores in essential oil. FPLC and FT-IR fingerprint analysis revealed the presence of bioactive constituents like rutin, tannic acid, vanillic acid and ascorbic acid in the essential oil. A strong anti-inflammatory activity of oil was observed using fluorescent spectroscopy. An appreciable in vitro antibacterial activity against gram-negative bacteria like Acetobacter aceti and Pseudomonas aeruginosa was detected. The data provides the scientific support to the use of essential oil from Eucalyptus globules as a potent herbal source of bioactive compounds possessing natural antioxidant activities in food and pharmaceutical industries.