Long-term use of Cu-conraining fungicides in eitrus groves has resulted in Cu buildup in the soil, but the effects of Cu contamination on microbial properties of citrus grove soils remain poorly understood. The objective of this study was to investigate the influence of long-term application of Cu-containing fungicides on microbial biomass, and bacterial community structure and diversity in five representative commercial citrus groves (5, 21, 27, 36, and 43 yr of planting history, respectively) soils, with one adjacent pasture soil as a reference. With increasing planting time of citrus, Cu concentrations in the soils increased, while microbiological properties including microbial biomass carbon (Cmic), microbial quotient (MQ), and the diversity of operational taxonomic units based on denaturing gradient gel electrophoresis (DGGE) community fingerprinting decreased. Stepwise multiple regression analysis showed that Cmic, microbial quotient, and bacterial community diversity were affected by Cu concentration and other soil properties such as total N and available P, but the effect of Cu was dominant.These results indicate that long-term application of Cu-containing fungicides has adverse effects on microbial biomass and bacterial community diversity in citrus grove soils. Sequencing of partial I6S rRNA gene fragments revealed that a shift of total bacterial community composition occurred as a result of Cu contamination, and the soils of more severely Cu-polluted citrus groves were dominated by bacteria g-Protcobacterium, Acldobacteria, Firmicutes, and b-Proteobacterium, whereas certain strains of s-Proteobacterium, g-Proteobacterium, Firmicutes, and Cyanobacteria were the dominant bacteria in the young citrus grove soils.