Soil-borne fungi and pests present significant challenges to crop production, causing diseases and reducing yields. This study explores the potential of producing chitinase from tomato cell suspension cultures and its application in controlling soil-borne fungi and pests. Tomato cell suspension cultures were established from seeds, and different elicitors—including chitin, fungal debris, and fungal metabolites—were used to optimize chitinase production. The enzyme was purified, and its physicochemical properties were characterized. Its antifungal activity was tested against various soil-borne fungi, while termiticidal activity was evaluated using four bioassay methods. Additionally, the nematicidal activity of chitinase was assessed against different stages of root-knot nematodes. Results indicated that chitin media induced the highest chitinase production and cell growth compared to other elicitors. The purified chitinase had a molecular weight of 34 kDa, with a maximum velocity (Vmax) of 0.940 μg NAG/min and a Michaelis constant of 1.236 mg colloidal chitin/mL. The turnover number (Kcat) was 1.598 μg NAG/min/mol chitinase. The enzyme exhibited a broad range of activity and stability across varying pH levels and temperatures, as well as remarkable stability under different storage times and in the presence of metal ions. Tomato-derived chitinase demonstrated superior antifungal, nematicidal, and termiticidal activities compared to common chemical fungicides and pesticides. Chitinase was successfully produced from tomato cell suspension culture in this study, highlighting its potential as a biocontrol agent for sustainable pest and disease management in agriculture.