The effect of sublethal concentrations (below the recommended field doses) of propiconazole and tebuconazole on the amount of tri transcripts and accumulation of trichothecenes by three Fusarium graminearum isolates of 3ADON, 15ADON, and NIV chemotypes was examined on yeast extract sucrose agar (YES) medium. RT-qPCR analyses showed higher tri4, tri5, and tri11 transcript levels in cultures of all three F. graminearum isolates supplemented with sublethal concentrations of azoles as compared to those in nontreated control, although the fold changes in the amount of tri transcripts differed according to the type of azole used. Mycotoxin analysis revealed higher increase in trichothecene accumulation in most of the tebuconazole-treated samples of all chemotypes tested. A huge increase in all trichothecene compounds was revealed in samples of all F. graminearum isolates treated with 5 mg L−1 of tebuconazole. An inducing effect of azoles on trichothecene accumulation in the grain was confirmed in an in planta experiment; however, the results obtained were inconsistent. A higher amount of trichothecenes and fungal DNA was quantitated in two grain samples treated with sublethal propiconazole concentrations. In contrast, no significant increase in trichothecene levels was revealed in grain samples treated with sublethal concentrations of tebuconazole.