The aim of this paper is to identify and investigate an endophytic fungus (strain 28) that was isolated from Houttuynia cordataThunb, a famous and widely-used Traditional Chinese Medicine. Based on morphological methods and a phylogenetic analysis of ITS sequences, this strain was identified as Chaetomium globosum. An antifungal activity bioassay demonstrated that the crude ethyl acetate (EtOAc) extracts of strain 28 had a wide antifungal spectrum and strong antimicrobial activity, particularly against Exserohilum turcicum(Pass.) Leonard et Suggs, Botrytis cinerea persoonand Botrytis cinereaPers. ex Fr. Furthermore, the fermentation conditions, extraction method and the heat stability of antifungal substances from strain 28 were also studied. The results showed that optimal antifungal activity can be obtained with the following parameters: using potato dextrose broth (PDB) as the base culture medium, fermentation for 4–8d (initial pH: 7.5), followed by extraction with EtOAc. The extract was stable at temperatures up to 80°C. This is the first report on the isolation of endophytic C. globosumfrom H. cordatato identify potential alternative biocontrol agents that could provide new opportunities for practical applications involving H. cordata.