In order to explore the possibility to identify common wound infection bacteria in mixed culture with gas chromatograph-ion migration spectroscopy (GC-IMS), the headspace gas of single and mixed cultures of Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa were detected and analyzed by GC-IMS system. The bacteria were cultured in thioglycolate medium tubes then transferred to the sampling bottles (indirect method), or directly cultured in the sampling bottles (direct method) to allow accumulation of volatile compounds and facilitate automation. The specific microorganism volatile organic compounds (mVOCs) of the three bacteria were obtained. Some of them have been known to certain substance, for example, ethanol, isoamyl acetate, Phenylacetaldehyde, 2-heptanone etc., while others have not. Principal component analysis (PCA) showed that a higher separability can be achieved with direct method than indirect method. This work indicated that it is possible to identify compound bacteria by detecting specific mVOCs with GC-IMS, and the specific mVOCs should be medium-dependent.