Significance and Impact of the Study: In this work, 143 endophytic fungi isolates belonging to 29 genera were obtained from two Rubia plants. Among them, four endophytic fungal strains were found to produce anti-tumour Rubiaceae-type cyclopeptides, which were main anti-tumour ingredients of Rubia plants. This is the first study on the systematic isolation of endophytic fungi from Rubia plants, and we believe that our work could provide valuable knowledges of endophytic fungi microbiome in Rubia plants for agricultural and industrial applications. Furthermore, our work could also exploit a new environmental-friendly resource of Rubiaceae-type cyclopeptides, which will avoid wildcrafting and overcollection of Rubia plants for environment protection.