Malignant mesothelioma (MM) is an incurable cancer. MM is often misdiagnosed, with a poor 5-year survival and limited treatment options. The discovery of endogenous volatile organic compounds (VOCs) is required in order to accelerate the development of a breath test as an alternative to conventional MM diagnosis. For the first time, this study used solid-phase microextraction and gas chromatography-mass spectrometry to identify VOCs released directly from the biphasic MM cell line MSTO-211H and the epithelioid MM cell line NCI-H28 as well as the non-malignant mesothelial cell line MET-5A. Multivariate statistical analysis showed separation between MSTO-211H, NCI-H28 and MET-5A results. 2-ethyl-1-hexanol was significantly increased in both MSTO-211H and NCI-H28 cells compared to MET-5A controls. In addition, ethyl propionate and cyclohexanol were significantly increased in MSTO-211H cells and dodecane was significantly increased in NCI-H28 cells. This is the first study reporting headspace analysis of these MM cell lines and the first to consider the effects of mesothelioma sub-type on VOC profile. Current results further highlight the potential for a diagnostic mesothelioma breath test as well as providing proof of concept for the differentiation between biphasic and epithelioid mesothelioma based on VOC profiles.