Aims A novel method has been developed that allows successful differentiation between Clostridium difficile culture-positive and culture-negative stool samples based on volatile organic compound (VOC) evolution and detection by headspace solid-phase microextraction coupled with gas chromatography mass spectrometry (HS-SPME-GC-MS). Methods and Results The method is based on the activation of p-hydroxyphenylacetate decarboxylase produced by Cl.difficile and the detection of a specific VOC, that is 2-fluoro-4-methylphenol from an enzyme substrate. In addition, other VOCs were good indicators for Cl.difficile, that is isocaproic acid and p-cresol, although they could not be used alone for identification purposes. One hundred stool samples were tested, of which 77 were positive by culture. Detection using HS-SPME-GC-MS allowed confirmation of the presence of Cl.difficile within 18h with a sensitivity and specificity of 83 center dot 1 and 100%, respectively. Conclusions It is recommended that this new approach could be used alongside conventional methods for Cl.difficile detection, including toxin detection methods, which would allow any false-negative results to be eliminated. Significance and Impact of the Study The ability to identify Cl.difficile-positive stool samples by the analysis of VOCs could allow the development of a VOC detection device which could allow rapid diagnosis of disease and hence prompt treatment with appropriate antibiotics.