The impact of herbicide applications during osmotic stress on plant metabolism is not yet well understood. In this study, the relationship between paraquat-induced oxidative stress (PQ) and polyethylene glycol-induced osmotic stress (PEG) was evaluated in terms of changes in proline and polyamine metabolisms. Metabolic changes were investigated in maize seedlings treated with PQ and PEG separately and PQ in combination with PEG. Relative to all treatments, the combination of PQ and PEG further increased damage to the membranes, hydrogen peroxide and proline content, reducing the water potential of the leaves. The PEG treatment significantly increased the putrescine and spermidine contents compared to all treatments, but did not significantly change the spermine content compared to controls and combinations. The expression of gene encoding pyrroline-5-carboxylate synthase increased after PQ treatment in combination with PEG compared to the singular stresses, while the expression of gene encoding proline dehydrogenase decreased. Gene expression levels of arginine decarboxylase and S-adenosylmethionine decarboxylase, responsible for polyamine biosynthesis, were highest in PEG-treated seedlings. Furthermore, the gene expression levels of polyamine oxidase and copper amine oxidase, which cause polyamine degradation, decreased in all treatments compared to the control. In conclusion, paraquat applied during osmotic stress may increase the H2O2 content and, therefore, further damage the maize seedling membrane. Increased membrane damage and production of H2O2 may play a role in the induction of genes coding for key enzymes in proline and polyamine metabolism pathways.