The goal of the present work was to identify the most important physiological and biological effects of titanium dioxide nanoparticles (TiO2 NPs) at a size of 83 ± 15 nm on plant parts. This was done by studying TiO2 physical and chemical properties through X-ray diffraction, scanning electron microscopy, Fourier transform infrared spectroscopy (FTIR), and Dynamic Light Scattering, and how TiO2 NPs move in plant organs and in the most important plant cells. Fenugreek (Trigonella foenum graecum L.) exposed to 100 mg L−1 of TiO2 NPs increased chlorophyll-a and -b and polyphenol contents, and decreased flavonoid level of leaves. Nano-stressed leaves and stems, therefore, displayed substantially increased catalase and ascorbate peroxidase activities. On the contrary, guaiacol peroxidase activities in the leaves and stems exposed to TiO2 NPs were significantly reduced. The result after 16 days of exposure to metal oxide was a substantial increase in the levels of nicotinamide adenine dinucleotide oxidase (NADH) in the leaves but a decrease in lipid peroxidation in the stems. The effects caused by exposure to NPs are regulated by specific isoforms of each compartment and each organ. TiO2 NPs were accumulated in the cell wall, resulting in the closure of plant cell pores that finished with the suspension of functions of the most important organelles targets such as mitochondria. We suggest that nanoparticles with size higher than 83 nm are transported via apoplastic pathway. Based on the correlations between mitochondria and cell apoptosis, accumulation of TiO2 in the cell wall causes lipid peroxidation and cell death. Metabolites changes in plants exposed to nano stressors have been determined by FTIR microspectroscopy. Environmental effect and risk of nanomaterial contamination have been studied after analyzing antioxidant enzymes. The role of oxidative stress markers in plant responses was well established.