High temperatures and the seasonality in tropical ecosystems favours plant pathogens, which result in many fungal diseases. Among these, diseases caused by Botryosphaeriaceae species are prominent as dieback, canker and leaf spots. In this research, we isolated one leaf-spot-causing Botryosphaeriaceae species from Ficus altissima leaves, which were collected in Guangzhou, Guangdong Province, China. Isolation and identification of the pathogen were based on morphological and molecular aspects. Based on multigene phylogenetic analysis of combined internal transcribed spacer (ITS), translation elongation factor 1-α gene (tef1) and beta-tubulin gene (tub2), the fungus associated with leaf spots on F. altissima is described as Lasiodiplodia fici, a novel species. Pathogenicity assays were conducted by inoculating the fungus onto detached shoots and plants under controlled environmental conditions. The results revealed that the L. fici isolates can infect the plant tissues under stress conditions by developing disease symptoms on detached shoots within three days. However, when it was inoculated onto the leaves of the host and grown in natural conditions, the progression of the disease was slow. The putative pathogen was re-isolated, and Koch’s assumptions were satisfied. This is the first report of Lasiodiplodia species causing disease on Ficus altissima. Results from the present study will provide additional knowledge on fungal pathogens associated with forest and ornamental plant species.