To get a better knowledge of the effects of residual chlorothalonil on soil characteristics and soil microbial communities, we evaluated the dissipation of chlorothalonil and the effects of different chlorothalonil concentrations on soil respiration, enzyme activities, and microbial community structure in yellow-brown loam soils. Bacterial and fungal soil communities were examined using traditional plate counting and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR–DGGE) methods. Soil properties and the results of DGGE band analysis were both used to estimate the status of the soil microbial ecosystem. The results show that residual chlorothalonil has considerable effects on soil respiration, enzymatic activities, and microbial community structure. In particular, soil respiration and phosphatase activities were increased, while saccharase activity, microbial biomass, and microbial community diversity were decreased by increasing levels of chlorothalonil treatment. Correlation analyses revealed that the application of chlorothalonil was significantly correlated with the change of the soil respiration, urease activity, sucrase activity, soil culturable bacteria and culturable fungi biomass. We conclude that residual chlorothalonil is directly related to soil respiration, enzyme activities, and microbial community structure.