In addition to ergosterol (I), 3b, 5a, 6b-trihydroxyergosta-7, 22-diene (II), 3b-hydroxy-ergosta-5-ene (Ⅲ), 3-oxo-ergosta-4, 6, 8 (14), 22-tetraene (IV), 3b-hydroxy-5a, 8a-epidioxy-ergosta-6, 22-diene (V), 3b-hydroxy-5a,8a-epidioxy-ergosta-6, 9 (11), 22-triene (VI) and 3-oxo-ergosta-4-ene (VII), a plant hormone indole-3-acetic acid (IAA) and three new antimicrobial metabolites were characterized from the culture of Colletotrichum sp., an endophyte isolated from inside the stem of Artemisia annua. The structures of the new metabolites were elucidated by a combination of spectroscopic methods (IR, MS, 1H and 13C NMR) as 6-isoprenylindole-3-carboxylic acid (1), 3b,5a-dihydroxy-6b-acetoxy-ergosta-7, 22-diene (2) and 3b, 5a-dihydroxy-6b-phenylacetyloxy-ergosta-7, 22-diene (3), respectively. The compounds 1-3 and Ⅲ-V inhibited the growth of all the tested bacteria (Bacillus subtilis, Staphylococcus aureus, Sarcina lutea and Pseudomonas sp.) with minimal inhibitory concentrations (MICs) ranging from 25 to 75 mg: ml. Moreover, metabolites 2 and 3, together with the known sterols III and V, were inhibitory against the fungi Candida albicans and Aspergillus niger with MICs between 50 and 100 mg: ml. At 200 mg: ml, compounds 1-3, III and IV were shown to be fungistatic to the crop pathogenic fungi Gaeumannomyces graminis var. tritici, Rhizoctonia cerealis, Helminthosporium sati6um and Phytophthora capisici. This is the first report on the endophytic fungus from A. annua and the bioactive metabolites thereof.