One hundred and five endophytic actinomycetes were isolated using four different media from surface-sterilized tissues of 7 plants samples of Alstonia scholar collected from Xishuangbanna Yunnan province, southeast China. The results showed that they belong to 9 genera, 7 families by 16S rRNA gene sequences analysis. All actinomycete strains fermentation liquids were carried out the antimicrobial activities test against five plant pathogenic fungi. The results showed that 12.4%, 14.3%, 11.4%, 12.4%, 8.6% endophytic actinomycetes presented antimicrobial activity for Fusarium graminearum, Phytophthora nicotianae, Alternaria alternata, Colletotrichum gloeosporioides and Candida albicans, respectively. Three strains with strong antibacterial activities were refermented and rescreened, their antimicrobial activities were stability, and may produce alkaloids.