To study the antimicrobial potential of vapor-phase Eucalyptus essential oil, the antimicrobial activity and minimum inhibitory concentrations (MIC) were evaluated against the tested microorganisms in vapor, liquid and solid phases. The chemical constitutes of Eucalyptus essential oil and its vapor were analyzed by gas chromatography-mass spectrometry (GC-MS) and solid phase microextraction (SPME) GC-MS. The results demonstrated that Eucalyptus essential oil vapor had obvious antimicrobial effect on the tested microorganisms. The antimicrobial activities of Eucalyptus essential oil vapor against Escherichia coli and Staphylococcus aurous were stronger than that against Aspergillus niger. The MIC value (0.045～0.36 mg/mL) in the vapor phase to the tested microorganisms was significantly lower than that in the liquid-phase MIC (4.5～18.0 mg/mL) and the solid phase MIC (4.5～18.0 mg/mL). The dominant components, mainly terpenes and alcohols, showed not significant difference between the Eucalyptus essential oil and its vapor. The antibacterial activity of Eucalyptus essential oil vapor was related to α-pinene, D-Limonene, 3-Carene, Terpinolene and Myrcene, and the relative increase in terpene contents led to the stronger antibacterial activity.