In this study, we evaluated bioinspired titanium dioxide nanoparticles (TiO2 NPs) that elicited biochemical and proteome modifications in wheat plants under the biotic stress caused by Puccinia striiformis f. sp. tritici (Pst). Biosynthesis of TiO2 NPs was confirmed using UV–Vis spectrophotometry, energy dispersive X-ray spectroscopy (EDX), scanning electron microscopy (SEM), and Fourier transform infrared (FTIR) spectroscopy. We found that the nanoparticles with crystalline nature were smaller than 100 nm. The results of FTIR analysis showed the presence of potential functional groups exhibiting O-H, N-H, C-C, and Ti-O stretching. The TiO2 NPs of different concentrations (20, 40, 60, and 80 mg L−1) were exogenously applied to wheat plants under the biotic stress caused by Pst, which is responsible for yellow stripe rust disease. The results of the assessment of disease incidence and percent disease index displayed time- and dose-dependent responses. The 40 mg L−1 TiO2 NPs were the most effective in decreasing disease severity. The bioinspired TiO2 NPs were also evaluated for enzymatic (superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT)), and nonenzymatic metabolites (total proline, phenolic, and flavonoid contents) in wheat plants under stripe rust stress. The 40 mg L−1 TiO2 NPs were effective in eliciting biochemical modifications to reduce biotic stress. We further evaluated the effects of TiO2 NPs through gel- and label-free liquid chromatography-mass spectrometry (LC-MS) proteome analysis. We performed proteome analysis of infected wheat leaves and leaves treated with 40 mg L−1 TiO2 NPs under stripe rust stress. The functional classification of the proteins showed downregulation of proteins related to protein and carbohydrate metabolism, as well as of photosynthesis in plants under biotic stress. An upregulation of stress-related proteins was observed, including the defense mechanisms and primary metabolic pathways in plants treated with 40 mg L−1 TiO2 NPs under stress. The experimental results showed the potential of applying biogenic TiO2 NPs to combat fungal diseases of wheat plants and provided insight into the protein expression of plants in response to biotic stress.