Listeria monocytogenes is a Gram-positive bacterium and an opportunistic food-borne pathogen which poses significant risk to the immune-compromised and pregnant due to the increased likelihood of acquiring infection and potential transmission of infection to the unborn child. Conventional methods of analysis suffer from either long turn-around times or lack the ability to discriminate between Listeria spp. reliably. This paper investigates an alternative method of detecting Listeria spp. using two novel enzyme substrates that liberate exogenous volatile organic compounds in the presence of α-mannosidase and d-alanyl aminopeptidase. The discriminating capabilities of this approach for identifying L. monocytogenes from other species of Listeria are investigated. The liberated volatile organic compounds (VOCs) are detected using an automated analytical technique based on static headspace–multi-capillary column–gas chromatography–ion mobility spectrometry (SHS–MCC–GC–IMS). The results obtained by SHS–MCC–GC–IMS are compared with those obtained by the more conventional analytical technique of headspace–solid phase microextraction–gas chromatography–mass spectrometry (HS–SPME–GC–MS). The results found that it was possible to differentiate between L. monocytogenes and L. ivanovii, based on their VOC response from α-mannosidase activity.