The contamination of barley by molds on the field or in storage leads to the spoilage of grain and the production of mycotoxins, which causes major economic losses in malting facilities and breweries. Therefore, on-site detection of hidden fungus contaminations in grain storages based on the detection of volatile marker compounds is of high interest. In this work, the volatile metabolites of 10 different fungus species are identified by gas chromatography (GC) combined with two complementary mass spectrometric methods, namely, electron impact (EI) and chemical ionization at atmospheric pressure (APCI)-mass spectrometry (MS). The APCI source utilizes soft X-radiation, which enables the selective protonation of the volatile metabolites largely without side reactions. Nearly 80 volatile or semivolatile compounds from different substance classes, namely, alcohols, aldehydes, ketones, carboxylic acids, esters, substituted aromatic compounds, alkenes, terpenes, oxidized terpenes, sesquiterpenes, and oxidized sesquiterpenes, could be identified. The profiles of volatile and semivolatile metabolites of the different fungus species are characteristic of them and allow their safe differentiation. The application of the same GC parameters and APCI source allows a simple method transfer from MS to ion mobility spectrometry (IMS), which permits on-site analyses of grain stores. Characterization of IMS yields limits of detection very similar to those of APCI-MS. Accordingly, more than 90% of the volatile metabolites found by APCI-MS were also detected in IMS. In addition to different fungus genera, different species of one fungus genus could also be differentiated by GC-IMS.