Objective:To explore a new natural antibiotic. Methods:The chemical composition of the essential oil from Ganoderma japonicum (G. japonicum) mycelia was analyzed by gas chromatography-mass spectrometry(GC-MS). The antimicrobial activity of the oil was evaluated against eighteen microorganisms, including bacteria, mildew and yeast by using a disc diffusion method. Furthermore, the minimum inhibitory concentrations(MIC) and the minimum bactericidal concentrations(MBC) of the essential oil against twelve clinical pathogens were determined. Results:The main components of the oil were nerolidol, decadienal, linalool and benzyl alcohol. The antimicrobial results indicated that the oil inhibited all the tested bacterium, especially Methicillin-resistant Staphylococcus aureus (MRSA) in which the antibacterial activity exhibited a MBC of 1.03 mg/ml. Conclusion: The essential oil of G. japonicum mycelium has significant inhibitory activity. It is a potential medicinal resource that can be used as a natural antibiotic.