Concerns about the widespread use of pesticides have been growing due to the adverse effects of chemicals on the environment and human health. It has prompted worldwide research into the development of a replacement to chemical disinfection of soil. The efficiency of steam sterilization, an alternative to chemical methods, has improved as technology has advanced, and the Agricultural Research and Extension Service in Korea recommends the use of steam sterilization. However, few studies have been conducted on the effects and operating conditions of high-temperature steam disinfection. In this study, we present the optimum operating conditions of a high-steam disinfector, to maximize the cost-effectiveness and removal efficiency of total nematodes and total bacteria in soil using the Box−Behnken design. The experimental data were fitted to a second-order polynomial equation using multiple regression analysis, with coefficients of determination (R2) for each model of 0.9279, 0.9678, and 0.9979. The optimum conditions were found to be a steam temperature of 150.56 °C, running speed of 1.69 m/min, and spray depth of 15.0 cm, with a corresponding desirability value of 0.8367. In the model, these conditions cause the prediction of the following responses: nematode removal efficiency of 93.99%, bacteria removal efficiency of 97.49%, and oil consumption of 70.49 mL/m2. At the optimum conditions for the steam disinfector, the removal efficiencies of nematodes and bacteria were maximized, and the oil consumption was minimized. The results of our study can be used as basic data for efficient soil disinfection using high-temperature steam.