Endophytic fungi are a remarkably diverse group of microorganisms that have imperceptible associations with their hosts for at least a part of their life cycle. The enormous biological diversity and the capability of producing bioactive secondary metabolites such as alkaloids, terpenoids, and polyketides have attracted the attention of different scientific communities, resulting in numerous investigations on these fungal endophytes. During our surveys of plant-root-based fungi in the mountain areas of Qingzhen, Guizhou Province, several isolates of endophytic fungi were identified. In this study, a novel endophytic fungus was discovered in the roots of a medicinal plant (Orixa japonica) in Southern China and introduced as a new species (Amphisphaeria orixae) based on morphological evidence and molecular phylogenetic analysis (combined ITS and LSU sequence data). To the best of our knowledge,A. orixaeis the first reported endophyte as well as the first hyphomycetous asexual morph inAmphisphaeria. A new isocoumarin, (R)-4,6,8-trihydroxy-5-methylisochroman-1-one (1), and 12 known compounds (2–13) were isolated from the rice fermentation products of this fungus. Using 1D- and 2D-NMR, mass spectrometry, and ECD studies, their structures were identified. The antitumor activity of these compounds was tested. Unfortunately, none of the compounds tested showed significant antitumor activity.