Pine Wilt Disease (PWD) has a significant impact on Eurasia pine forests. The microbiome of the nematode (the primary cause of the disease), its insect vector, and the host tree may be relevant for the disease mechanism. The aim of this study was to characterize these microbiomes, from three PWD-affected areas in Portugal, using Denaturing Gradient Gel Electrophoresis, 16S rRNA gene pyrosequencing, and a functional inference-based approach (PICRUSt). The bacterial community structure of the nematode was significantly different from the infected trees but closely related to the insect vector, supporting the hypothesis that the nematode microbiome might be in part inherited from the insect. Sampling location influenced mostly the tree microbiome (P < 0.05). Genes related both with plant growth promotion and phytopathogenicity were predicted for the tree microbiome. Xenobiotic degradation functions were predicted in the nematode and insect microbiomes. Phytotoxin biosynthesis was also predicted for the nematode microbiome, supporting the theory of a direct contribution of the microbiome to tree-wilting. This is the first study that simultaneously characterized the nematode, tree and insect-vector microbiomes from the same affected areas, and overall the results support the hypothesis that the PWD microbiome plays an important role in the disease's development.