The aim of the study was to determine the effect of fungicidal treatment and storage on the occurrence of mycotoxins in barley (Hordeum vulgare L.). Barley was initially inoculated with Fusarium culmorum followed by the application of fungicides (prothioconazole and bixafen). A screening of 57 mycotoxins were performed using ultra-performance liquid chromatography in tandem with mass spectrometry. The fungicide treatment affected (P <0.05) the levels of zearalenone, β-zearalenol, arternariol and alternariol-methylether that were present. Levels of deoxynivalenol was highest in the second year of monitoring. 3-acetyl-deoxynivalenol was not affected by fungicidal treatment or storage. The significant increase (P <0.05) of DON-3-glucoside, 15-acetyl-DON, enniatin A, enniatin A1, enniatin B, and enniatin B1 was measured in barley samples. The results of the experiment determined that the use of fungicides can suppress some kinds of mycotoxins, but not others.