Durum wheat grain can accumulate mycotoxins because it is highly sensitive to infections caused by pathogens of the genera Fusarium and Alternaria. Reduced fungicide use increases the demand for biological methods of pathogen control. The aim of the experiment was to evaluate the efficacy of Debaryomyces hansenii (Dh) yeast in reducing the content of secondary fungal metabolites present in the spikes of five durum wheat cultivars grown in southern and northern Poland. A total of 27 Fusarium metabolites and nine metabolites produced by other fungi were identified in the grain. The application of the Dh yeast strain decreased deoxynivalenol concentration in all samples relative to control treatments (by 14–100%) and treatments inoculated with F. graminearum (by 23–100%). In northern Poland, the biological treatment also led to a considerable reduction in the content of culmorin (by 83.2–100%) and enniatins A1 and B (by 9.5–65.3% and 6.7–70%, respectively) in the grain. An analysis of multiple fungal metabolites is a highly useful tool for determining grain quality and its suitability for consumption. When applied in the flowering stage, yeasts can partly complete fungicides in reducing Fusarium head blight.