The main aim of this research work was extraction, characterization and optimization of essential oil from pumpkin seed, cucurbita pepo variety using ethanol as a solvent. The extraction was carried out by using a Soxhlet, leaching method and oil was separated from solvents by using rotary evaporator. Pumpkin seed was analyzed for proximate analysis and found that moisture content of 5.21 ± 0.12%, crude proteins content of 22.5±0.31%, crude fat content of 50.5±0.112%, crude fiber content of 7±0.09% ash content of 4.5±0.19%, and carbohydrate content of 10.37±0.079%. A full factorial experimental randomized design with three factors two levels with triplicate six center points with a total of 30 experimental runs was carried out to optimize the operational process factors on the yield of essential oil. The factors and their operating ranges were: extraction time from 2 to 6 hours, particle size from 0.25 mm to 2mm, and solvent to pumpkin seed meal ratio of 0.05mg/ml to 0.1 mg/ml under constant temperature of 80℃. Physic-chemical properties of extracted essential oil analyzed and found that specific gravity of 0.91178, pH of 5.16±0.1kinematic viscosity 35, density 911.78, moisture content 0.07%, volatile contents 1.468, refractive index 0.62±0.61, free fatty acid 97.5I2g/100g, saponification value of 1.23±0.23mg/KOH/g oil and 189.8±0.31mgKOH/g oil, which is in close agreement with the literature values. The FT-IR analysis shown that the presence of functional group of carbohydrate, carbonyl, alkene, aromatics, alkane, aliphatic amine, carboxylic acid, alcohol and methyl ester which is closest to essential oil composition in the literature. Gas-Chromatography-Mass Spectroscopy analysis shown that the presence of five major free fatty acids: linoleic acid of 57.5%, oleic acid of 20.2%, palmitic acid 19.1%, stearic acid 2.97%, and miystiric acid 0.23%. The pumpkin seed essential oil also showed better antimicrobial activity against both S. aureus and E. coli with maximum zone of inhibition 15.5±0.23 mm and 13.5±0.51 mm, respectively. Therefore, pumpkin seed essential oil, it is feasible to be used as antimicrobial and other applications.