Nitrogen fertilization combined with microelements is an effective way to provide nutrients to plants, which are essential for obtaining high-value crops. The aim of this study was to evaluate two N fertilizer rates (150 and 200 kg ha-1) and four Mn fertilizer rates (0.0, 0.5, 1.0, and 1.5 kg Mn ha-1) on the N, Mn, Zn, Cu, and Fe content and composition of fatty acids in winter wheat (Triticum aestivum L.) grain. An increase in the N fertilizer rate increased (12.7%) the Mn content and decreased (10%) the Cu content of wheat grain. Regardless of N fertilization, foliar application of Mn at 1.5 kg ha-1 contributed to the highest Zn (28.4 mg kg-1) and Fe (58.4 mg kg-1) content in the grain. In an analysis of lipid fractions, the highest value of the coefficient of variation was recorded for C18:0 (16.3%-low variation). Nitrogen and Mn fertilization were most strongly correlated with the Mn content of grain (r = 0.356, r = 0.391, respectively). The 200 kg N ha-1 treatments combined with 1.0 kg ha-1 Mn and 150 kg ha-1 N without Mn were correlated with the content of C:18:0, C18:1c11, C18:1c9, and monounsaturated fatty acids in the grain. The application of 200 kg ha-1 N with 1.5 kg ha-1 Mn was correlated with the Fe, Zn, and Mn content of the grain. The remaining fertilization treatments were correlated with the content of C18:3, C18:2, polyunsaturated fatty acids (PUFA), and the C18:2/C18:3 ratio in the grain. Results indicated that the application of 200 kg ha-1 N beneficially affected the PUFA content in the winter wheat grain and can therefore be used to obtain raw material with increased nutritional value. The human organism does not synthesize PUFA, so they must be taken with food (or supplements); winter wheat grain can be a good source because it contains more than 60% PUFA.