Fusarium solani is a plant pathogenic fungus that causes tomato root rot disease and yield losses in tomato production. The current study’s main goal is testing the antibacterial efficacy of chitosan nanoparticles loaded with Thyme vulgaris essential oil (ThE-CsNPs) against F. solani in vitro and in vivo. GC-MS analysis was used to determine the chemical constituents of thyme EO. ThE-CsNPs were investigated using transmission electron microscopy before being physicochemically characterized using FT-IR. ThE-CsNPs were tested for antifungal activity against F. solani mycelial growth in vitro. A pot trial was conducted to determine the most effective dose of ThE-CsNPs on the morph/physiological characteristics of Solanum lycopersicum, as well as the severity of fusarium root rot. The relative gene expression of WRKY transcript factors and defense-associated genes were quantified in root tissues under all treatment conditions. In vitro results revealed that ThE-CsNPs (1%) had potent antifungal efficacy against F. solani radial mycelium growth. The expression of three WRKY transcription factors and three tomato defense-related genes was upregulated. Total phenolic, flavonoid content, and antioxidant enzyme activity were all increased. The outfindings of this study strongly suggested the use of ThE-CsNPs in controlling fusarium root rot on tomatoes; however, other experiments remain necessary before they are recommended.