Leaf spot of soapberry, Sapindus mukorossi is a disease new to China. The disease significantly reduces not only the plant’s ornamental value but also its medicinal properties. Identification of pathogens timely assists preventing and controlling this disease. In July 2019, foliage of soapberry on the campus of Nanjing Forestry University, China, was infected by the disease, and the disease incidence was 96%. Symptomatic leaves were collected from three infected trees. After isolating the fungus, its pathogenicity was tested to satisfy Koch’s postulates, and the culture was identified based on its morphological features and multi-gene phylogenetic analyses. Isolates WHZ3 and YB1 from the diseased leaves were identified as Diaporthe biconispora, and the identification was confirmed using morphological features and multi-gene phylogenetic analyses derived from an internal transcribed spacer (ITS), elongation factor 1-alpha (EF1-α), beta-tubulin (β-tub), histone H3 (HIS), and calmodulin (CAL). Koch’s postulates proved the fungus to be a pathogen on soapberry. Diaporthe biconispora was previously reported as an endophyte in plants of the Citrus group, but was shown to be pathogenic to soapberry causing leaf spots.