Abstract: To identify the antagonistic bacteria of Gonatophragmium, this study investigated variations in the diversity, structure, and function of phylloplane microorganisms in the mulberry ring rot disease areas of mulberry canopy. Using high-throughput sequencing technology, we analyzed the relative abundance of bacteria on the upper and lower surfaces of the upper and lower leaves in the canopy. The results showed that 1) the Shannon index in upper canopy was 1.26 times of that in the lower canopy, and the upper surface of leaves was 1.49 times of that in the lower surface. 2) The dominant genera in the lower surface of a leaf in the lower canopy (LB) and the lower surface of a leaf in the upper canopy (UB) were the genus Pantoea, with relative abundances of 38.04% and 25.31%, respectively. In the upper surface of a leaf in the lower canopy (LS), it was Serratia and the upper surface of a leaf in the upper canopy (US) was Stenotrophomonas, with relative abundances of 18.0% and 23.73%, respectively. 3) The transport and metabolism of carbohydrates and amino acids of bacteria in the lower canopy leaves were stronger than those in the upper canopy leaves; however, lipid metabolism and transport were weaker in the lower canopy leaves. The cell wall biogenesis function of bacteria on the upper surface of leaf in upper canopy was stronger than that on the lower surface, whereas the amino acid transport and metabolism functions were weaker on the upper surface. The biosynthesis, transport, and catabolism of secondary metabolites, lipid transport, and the metabolism of bacteria on the upper surface of lower canopy leaves were stronger than those on the lower surface, but cell movement was weaker on the upper surface. The relative abundances of Bacillus, Asticcacaulis, and Phenylobacterium were significantly negatively related to the relative abundance of pathogens (P < 0.05); the most significant correlation was for Bacillus (-0.87). These results indicate significant differences in the diversity, structure, and function of bacterial communities on the upper and lower surfaces of mulberry leaves in the canopy, which will assist further research on the biological control of mulberry ring rot disease.