Here, TiO2 nanoparticles (NPs), with rod-like morphology, were synthesised under hydrothermal conditions as a green and efficient nanopesticide against bacterial wilt Ralstonia solanacearum. The NPs had a rutile crystal structure with an average particle size of 67.60 nm. FeCl3 was used as a crystal modification controlling agent to make the nanorods well dispersed. In vitro antibacterial experiments indicated that 0.01–0.1 mg/mL TiO2 NPs exhibited high bioactivity toward R. solanacearum at different time intervals (6 h, 12 h, and 24 h), and the inhibition activity of TiO2 NPs increased with concentration. TiO2 NPs worked by inducing the disorganisation and impairing the integrity of cell membranes. The degree of damage correlated to the concentration of TiO2 NPs. Furthermore, exposure to TiO2 NPs induced severe genomic DNA injury to the bacteria, decreased disease-causing gene (phcB, phcR, epsA, epsF, egl, pilT and hrp) expression, and reduced levels of cellulose and pectinase. All these destructive effects might be due to the intracellular production of reactive oxygen species (1O2, •O2- and •OH) which was stimulated by TiO2 NPs. These observations provide new insights into the antibacterial mechanisms of intracellular ROS peroxidation damage. Additionally, 9-week-old tomato plants were exposed to different doses of nano-TiO2 (0, 0.01, 0.05, and 0.1 mg/mL) for 24 h. Using SEM analysis and the contact angles test, it was found that TiO2 NPs could attach to the surface of tomato leaves. The sprayed TiO2 NPs could be transported and bioaccumulated in the leaf, stem, and root tissues of tomato, and the maximum concentration of TiO2 NPs was observed in sprayed leaves at 7 days. Moreover, the detached tomato leaves and pot experiments revealed that TiO2 NPs could enhance the disease resistance of plants against tomato wilt, significantly increase the content of peroxidase (POD) and catalase (CAT), and had no obvious effect on plant growth. All these results indicated that TiO2 NPs can serve as an eco-friendly, biocompatible, and efficient nanopesticide against tomato wilt.