The paper shows the effect of nitrogen nutrition, exogenous auxin, and rhizosphere auxin-synthesizing microorganisms on the variability of the composition of fatty acids (FA) in the vegetative organs of spring wheat. The object of the study was seedlings of spring soft wheat Triticum aestivum L. The determination of FAs was carried out by gas chromatography with mass spectrometry (GC-MS). Analysis of FAs showed that in the control variants (without auxin), nitrogen nutrition conditions did not affect the localization of polyunsaturated FAs in vegetative organs; the maximum content of triene FAs was observed in leaves of 48.30% (N-deficient variant) and 44.8% (NO-3-variant ) and the absence of these FAs in the roots. It was found that in the presence of nitrates, the proportion of unsaturated FAs in the leaves and roots of wheat decreases. The use of exogenous auxin (5–50 μg/ml) in the early stages of ontogenesis leads to an increase in the amount of saturated (palmitic and stearic) acids and a decrease in unsaturated acids in vegetative organs, regardless of the conditions of nitrogen nutrition. During the introduction of spring wheat seedlings by auxin-synthesizing microorganisms, it was noted that nitrogen-fixing bacteria affect the leaves of plants most effectively, the content of saturated FAs increases by 72%, and only 16% increases in these FAs in the leaves of nitrate-reducing microorganisms.