The wild resources ofPsammosilene tunicoideshave decreased sharply because of the long-term mining and excavation, which has led to the increased demand for its artificial cultivation. However, root rot represents a significant obstacle leading to a poor quality and product ofP. tunicoides. Previous reports have not focused on root rot inP. tunicoides.Therefore, this study explores the rhizospheric and root endophytic microbial community structure and composition of healthy and root rotP. tunicoidesto understand the mechanism underlying root rot.The properties of the rhizosphere soil were assessed using physiochemical methods, and the bacterial and fungal populations were studied through amplicon sequencing of the 16S rRNA genes and ITS regions in the root and soil. Compared to healthy samples, the pH, hydrolysis N, available P, and available K were significantly decreased in the diseased samples while the organic matter and total organic carbon were significantly increased in the diseased samples. Redundancy analysis (RDA) showed that soil environmental factors are related to changes in the root and rhizosphere soil microbial community ofP. tunicoidesindicating that the physiochemical properties of soil affect plant health. Alpha diversity analysis showed that the microbial communities of healthy and diseased samples were similar. Some bacterial and fungal genera were significantly increased or decreased (P< 0.05) in diseasedP. tunicoides, and certain microbial factors that antagonized root rot were further explored. This study provides an abundant microbial resource for future studies and contributes to improving soil quality andP. tunicoidesagricultural production.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00284-023-03290-4.