Fusarium head blight and inadequate nitrogen fertilization can cause numerous biochemical changes in wheat. The main aim of this study was to determine the effects of Fusarium inoculation and a broader range of different nitrogen fertilization on the defense response in the spikes of four wheat varieties, varying in FHB susceptibility. Total soluble phenolics content, activities of enzymes involved in phenol metabolism (PAL and PPO), and total antioxidant capacity were determined as indicators of defense response. In both growing seasons, Fusarium inoculation altered PHE content in wheat spikes, indicating involvement of PHE in the defense response to Fusarium attack. Increased PHE content in the partially resistant varieties (Apache and Graindor) indicates involvement of PHE in the defense response and better disease tolerance in the more resistant varieties. Breeding wheat varieties with enhanced PHE synthesis could be a promising strategy to control FHB. To the best of our knowledge, this is the first study that emphasizes the effects of Fusarium infection and a broader range of different nitrogen fertilization on PHE and enzymes involved in PHE metabolism. In addition, this is the first study using the FRAP method to determine the antioxidant capacity of wheat tissues under the influence of Fusarium infection and different nitrogen fertilization.