Fusarium head blight caused by a complex of Fusarium species is widespread across the world and ranks among the most serious diseases in cereals. Long-term field experiments were set up to evaluate the effects of preceding crop and soil management methods on Fusarium mycotoxin (DON, deoxynivalenol) contamination of winter wheat and spring barley grain. Winter wheat and spring barley were cultivated at two locations in the Czech Republic (A: Ivanovice na Hané during 2002-2014, and B: Žabčice during 2007-2014) with preceding crops (A) alfalfa, maize, and pea; and (B) alfalfa (only for wheat), sugar beet (only for barley), and maize. Different soil management methods also were used: (A) 22 cm tillage, 15 cm tillage, 10 cm chisel, and direct drilling; and (B) 22 cm tillage, 10 cm chisel, and direct drilling. Mycotoxin content in harvested grain was analysed using ELISA. At both locations in the experiments with both wheat and barley, year had a significant effect on mycotoxin content in grain. Preceding crop was another significant factor in wheat experiments at both locations, with DON content in grain higher with maize as the preceding crop than in the cases of other preceding crops. Soil management method had a significant effect only on mycotoxin content in wheat grain grown at Žabčice, and the highest DON content was determined in the chisel variant, in which case a large amount of harvest residue remained on the soil surface or was only partially incorporated.