Abstract: In this study, microwave-assisted extraction was applied to extract the essential oil from Ocimum basilicum Linn with petroleum ether. By using orthogonal methodology, the effects of concentration of Na Cl, soaking time, solid-liquid ratio, microwave power and extraction time were investigated. It was found that when soaking time was 6 h, solid-liquid ratio was1∶ 16, microwave power was 567 W and microwave time was 120 s, the optimal oil yield was 0.3758% ( n = 3) . Then, essential oil's antibacterial effect was determined by using paper discassay and fold dilution plate method.The results indicated that the minimum inhibitory concentration ( MIC) for Escherichia coli, Bacillus subtilis and Staphylococcus aureus were 31.25, 62.5, 125 μg/m L, respectively, and the minimum bactericidal concentration ( MBC) for these were 31.25, 125, 500 μg/m L, respectively.The antibacterial order was: Escherichia coli > Bacillus subtilis > Staphylococcus aureus. The research provides the theoretical basis for development and comprehensive utilization of Ocimum basilicum Linn.