In order to explore the combined ecological effects of acetochlor and copper sulphate on soil microorganisms, the traditional toxicological methods and BIOLOG were employed. The results indicate that the combined application of acetochlor and copper sulphate has acute inhibitory effects on the amount of culture-dependent viable bacteria, actinomycetes and fungi as well as soil dehydrogenase activity. These effects are gradually weakened and even reverse with the increase of exposure time. The change of the substrate-induced respiration (SIR) is significantly increased under the combined stress. The data of BIOLOG are subjected to the principle component analysis (PCA) and three index models such as Shannon, Simpson and McIntosh indexes. The results also indicate that the combined application of acetochlor and copper sulphate can destroy the richness and evenness of microbial community diversity in phaeozem. The changes of carbon utilization diversity of phaeozem microbial community are also showed by the pattern of PCA.