Fusarium infection in wheat causes Fusarium head blight, resulting in yield losses and contamination of grains with trichothecenes. Some plant secondary metabolites inhibit accumulation of trichothecenes. Eighteen Fusarium infected wheat cultivars were harvested at five time points and analyzed for the trichothecene deoxynivalenol (DON) and 38 wheat secondary metabolites (benzoxazinoids, phenolic acids, carotenoids, and flavonoids). Multivariate analysis showed that harvest time strongly impacted the content of secondary metabolites, more distinctly for winter wheat than spring wheat. The benzoxazinoid 2-β-glucopyranoside-2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA-glc), α-tocopherol, and the flavonoids homoorientin and orientin were identified as potential inhibitors of DON accumulation. Several phenolic acids, lutein and β-carotene also affected DON accumulation, but the effect varied for the two wheat types. The results could form a basis for choosing wheat cultivars using metabolite profiling as a marker for selecting wheat cultivars with improved resistance against Fusarium head blight and accumulation of trichothecene toxins in wheat heads.