ABSTRACT: In this study, the occurrence of multiple fungal metabolites including mycotoxins was determined in four different winter wheat varieties in a field experiment in Croatia. One group was naturally infected, while the second group was inoculated with a Fusarium graminearum and F. culmorum mixture to simulate a worst-case infection scenario. Data on the multiple fungal metabolites including mycotoxins were acquired with liquid chromatography with mass spectrometry (LC-MS/MS) multi-(myco)toxin method. In total, 36 different fungal metabolites were quantified in this study: the Fusarium mycotoxins deoxynivalenol (DON), DON-3-glucoside (D3G), 3-acetyldeoxynivalenol (3-ADON), culmorin (CULM), 15-hydroxyculmorin, 5-hydroxyculmorin, aurofusarin, rubrofusarin, enniatin (Enn) A, Enn A1, Enn B, Enn B1, Enn B2, Enn B3, fumonisin B1, fumonisin B2, chrysogin, zearalenone (ZEN), moniliformin (MON), nivalenol (NIV), siccanol, equisetin, beauvericin (BEA), and antibiotic Y; the Alternaria mycotoxins alternariol, alternariolmethylether, altersetin, infectopyron, tentoxin, tenuazonic acid; the Aspergillus mycotoxin kojic acid; unspecific metabolites butenolid, brevianamid F, cyclo(L-Pro-L-Tyr), cyclo(L-Pro-L-Val), and tryptophol. The most abundant mycotoxins in the inoculated and naturally contaminated samples, respectively, were found to occur at the following average concentrations: DON (19,122/1504 µg/kg), CULM (6109/1010 µg/kg), 15-hydroxyculmorin (56,022/1301 µg/kg), 5-hydroxyculmorin (21,219/863 µg/kg), aurofusarin (43,496/1266 µg/kg). Compared to naturally-infected samples, Fusarium inoculations at the flowering stage increased the concentrations of all Fusarium mycotoxins, except enniatins and siccanol in Ficko, the Aspergillus metabolite kojic acid, the Alternaria mycotoxin altersetin, and unspecific metabolites brevianamid F, butenolid, cyclo(L-Pro-L-Tyr), and cyclo(L-Pro-L-Val). In contrast to these findings, because of possible antagonistic actions, Fusarium inoculation decreased the concentrations of the Alternaria toxins alternariol, alternariolmethylether, infectopyron, tentoxin, tenuazonic acid, as well as the concentration of the nonspecific metabolite tryptophol.