Isothiazolinones are a group of effective biocides, which are widely used in personal hygiene, pharmaceutical and water treatment products, but their application effects for soil sterilization are still unclear. In this paper, a commercially sold isothiazolinone biocides Kathon CG was tested for soil sterilization. The populations of bacteria, fungi and actinomycetes in tested soil were determined before and after isothiazolinone application at different concentrations, soil suspension pH values and temperatures for two durations of equilibration time. The results showed that the killing rates of actinomycetes in the tested soils were 100%, and the killing rates of bacteria and fungi in the tested soils increased with the increase of Kathon CG concentration. When Kathon CG concentration enhanced from 0.5 ml/L to 3.0 ml/L, the killing rate of bacteria and fungi in air-dried soil increased from 84.1% and 78.0% to 98.7% and 99.6%, respectively, the killing rate of bacteria and fungi was close to 100% when Kathon CG concentration reached to 5.0 ml/L or above. When the concentration of Kathon CG was 0.5 ml/L and 1.0 ml/L, the killing rates of bacteria and fungi for fresh soil declined compared that of the air-dried soil, and in this case, excessive alkaline soil suspension pH also reduced the killing rate of bacteria and fungi. When Kathon CG concentration reached to 3.0 ml/L or above, the killing rate of bacteria and fungi in air-dried soil and fresh soil was over 99.0%, and soil suspension pH had little effect on the killing rate of bacteria and fungi. The ambient temperature (25-50℃) had no influence on the germicidal effect of soil bacferia, and the duration of the bactericidal effect was longer than 72 h. This study shows that Kathon CG can effectively kill bacteria, fungi and actinomycetes in the soil at a low concentration. The germicidal effect remains stable at 25℃-50℃, less affected by pH of soil suspension. The use of Kathon CG may be one of the alternative methods in inhibiting soil microbial processes.