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The accidents of aniline spill and explosion happened almost every year in China, whereas the toxic effect of aniline on soil microbial activity remained largely unexplored. In this study, isothermal microcalorimetric technique, glucose analysis, and soil enzyme assay techniques were employed to investigate the toxic effect of aniline on microbial activity in Chinese soil for the first time. Soil samples were treated with aniline from 0 to 2.5 mg/g soil to tie in with the fact of aniline spill. Results from microcalorimetric analysis showed that the introduction of aniline had a significant adverse effect on soil microbial activity at the exposure concentrations ≥0.4 mg/g soil (p < 0.05) and ≥0.8 mg/g soil (p < 0.01), and the activity was totally inhibited when the concentration increased to 2.5 mg/g soil. The glucose analysis indicated that aniline significantly decreased the soil microbial respiratory activity at the concentrations ≥0.8 mg/g soil (p < 0.05) and ≥1.5 mg/g soil (p < 0.01). Soil enzyme activities for β-glucosidase, urease, acid-phosphatase, and dehydrogenase revealed that aniline had a significant effect (p < 0.05) on the nutrient cycling of C, N, and P as well as the oxidative capacity of soil microorganisms, respectively. All of these results showed an intensively toxic effect of aniline on soil microbial activity. The proposed methods can provide toxicological information of aniline to soil microbes from the metabolic and biochemical point of views which are consistent with and correlated to each other.