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Background: Panax notoginseng (Burkill) F. H. Chen is a Chinese medicinal plant of the Araliaceae family commonly used in the treatment of cardiovascular and cerebrovascular diseases in Asia and elsewhere. To meet an increase in Chinese herbal medicine market demand, most P. notoginseng is planted artificially, and is vulnerable to various plant diseases. Root rot disease, in particular, causes substantial P. notoginseng yield reduction and economic losses. High-depth next-generation sequencing technology was used to analyze the rhizosphere and root endophyte microbial communities of P. notoginseng to compare the characteristics of these two communities between healthy and root rot diseased P. notoginseng plants, and to clarify the relationship between these microbial communities and root rot disease.Results: The P. notoginseng rhizosphere microbial community was more diverse than the root endophyte community, and the difference in functional pathways between healthy and diseased P. notoginseng plants was greater in the root endophyte than in the rhizosphere communities. Multi-database annotation results showed that the highest number of endophytic bacteria occurred in the roots of diseased plants. The number of carbohydrate-active enzymes database families was also higher in diseased roots. The RND antibiotic efflux function was higher in the healthy samples. A high abundance of Variovorax paradoxus and Pseudomonas fluorescens occurred in the healthy and diseased root endophyte communities, respectively. Ilyonectria mors-panacis and Pseudopyrenochaeta lycopersici were most abundant in the diseased samples. In addition, the complete genome of two unknown Flavobacteriaceae species and one unknown Bacteroides species were obtained based on binning analysis.Conclusions: The rhizosphere and root endophyte microbial communities of healthy and root rot diseased P. notoginseng showed marked differences in diversity and functional pathways. The higher mapping values obtained for the diseased samples reflected the occurrence of root rot disease at the molecular level. Variovorax paradoxus and Pseudomonas fluorescens may be antagonistic bacteria of root rot in P. notoginseng, whereas Ilyonectria mors-panacis and Pseudopyrenochaeta lycopersici appear to be P. notoginseng root rot pathogens. Our study provides a theoretical basis for understanding the occurrence of root rot in P. notoginseng and for further research on potential biological control agents.