Nod factors among the signaling molecules produced by rhizobia in response to flavonoids to induce root nodule formation in the legumes. It is, however, hypothesized that they might increase the yield and positively impact the growth of non-legumes. To evaluate this statement, rapeseed treated with Nod factor-based biofertilizers were cultivated, their stems was collected, and the metabolic changes were investigated using Raman spectroscopy and MALDI mass spectrometry imaging. Biofertilizer proved to increase the concentration of lignin in the cortex, as well as hemicellulose, pectin, and cellulose in the pith. Moreover, the concentration of quercetin derivatives and kaempferol derivatives increased, while the concentration of isorhamnetin dihexoside decreased. The increase in the concentration of the structural components in the stem might therefore increase the lodging resistance, while the increase in concentration of the flavonoids might increase their resistance to fungal infection and herbivorous insects.