Barley can be contaminated with a wide range of fungal secondary metabolites, including various mycotoxins that reduce the quality and safety of raw materials as well as cause economic losses. A survey was conducted for the crop seasons 2016 and 2017 to analyse fungal metabolites, including mycotoxins, in grain and straw samples of barley, which originated from fields across Switzerland. In total, 253 grain and 237 straw samples were analysed by LC-MS/MS detecting 87 and 86 fungal metabolites, respectively, which are reported to be produced by Fusarium, Alternaria, Claviceps, Aspergillus, Penicillium and other genera. None of the grain samples exceeded the permitted limits of mycotoxins set by the European Commission. With regard to straw, three and six samples exceeded the guidance levels set for raw grains for deoxynivalenol and the sum of T-2 and HT-2, respectively. Nevertheless, some samples contained high concentrations of unregulated fungal metabolites, e.g. enniatins, infectopyron, zinniol and rubellin D. This was more frequently observed in straw and, to a lesser extent, in grains, suggesting that the presence of fungal metabolites in straw material should not be neglected. Our study demonstrated that both grain and straw matrices of barley represent large pools of various fungal secondary metabolites, most of them with undetermined toxicity. Hence, future studies should focus on the toxicology of the predominant fungal metabolites that occurred at elevated concentrations as well as the health impact of co-occurrence of toxins primarily with metabolites that revealed strong correlations.