In 2010 and 2011, studies to determine the optimal timing of prothioconazole application (200 g a.i./ha) for reducing Fusarium mycotoxin accumulation in grain were conducted in controlled replicated experiments under small-plot mist-irrigated experiments and in field-scale experiments using two hybrids susceptible to F. gramineaerum infection. A significant decrease in total deoxynivalenol (DON) [DON + 15-acetyl-DON + DON 3-glucoside + 3-acetyl-DON] and zearalenone concentrations was observed when fungicide was sprayed at VT (tasseling) and R1 (silking; P < 0.01) followed by applications at V18 (18th leaf) and R2 (blister; P < 0.05) stages, corresponding to silk completely emerged and fully elongated and to silk emergence and browning, respectively. No reduction in Fusarium graminearum toxins was found after silk senescence (R3 or milk) stage. Moniliformin, fumonisins, beauvericin, enniatins, HT-2 and T-2 toxins were also found in small quantities, and no reduction was observed after treatment (P > 0.05). Mean reduction (±s.d.) of 59 ± 20% and 57 ± 38% of total DON and zearalenone was observed at full silk elongation, respectively.