In this study, the diversity of endophytic fungi isolated from different tissues of healthy P. notoginseng was evaluated, and the isolates were screened for their potential antimicrobial activity against five major phytopathogens causing root rot of P. notoginseng. To the best of our knowledge, this is the first report of the biodiversity, phylogeny, and assessment of biocontrol potential of endophytic fungi harbored in P. notoginseng.