Twig blight is a serious disease of bayberry, which limits bayberry production. In order to prevent and manage the disease, we used high-throughput sequencing technology to analyze bacterial and fungal community richness and diversity in various organs of the tree, bulk and rhizosphere soil of healthy and diseased bayberry. The results showed significant differences in richness and diversity of bacteria and fungi in bulk soil, rhizosphere soil, roots, trunks, barks, and leaves between twig blight diseased trees and healthy trees. In bulk soil, the richness and diversity of bacteria significantly decreased, while that of fungi significantly increased. In barks of diseased trees, the richness and diversity of bacteria significantly increased, and those of fungi significantly decreased. The relative abundance of dominant bacteria and fungi in various organs, bulk soil, and root surface soil changed significantly at phylum, class, and genus levels in the diseased trees. The relative abundance of Pseudomonas sp. on the trunks, roots, and root surface soils of diseased trees significantly decreased, and Fusarium sp. of the diseased root surface and bulk soils also significantly decreased, while the relative abundance of Penicillium sp. on the diseased root surface and bulk soils significantly increased. Pestalotiopsis sp., from the same genus as the twig blight pathogen, was less abundant in the roots but more abundant in the leaves, trunks, barks as well as root surface soils and bulk soils of the diseased bayberry trees than those of the health trees. The relative abundance of Pestalotiopsis sp. was positively correlated with those of most of the fungi. Our results might provide useful theoretical basis for the development of ecological improvement and healthy-tree cultivation technology, and biological control of bayberry twig blight disease.