Species of Botryosphaeriaceae are important pathogens and endophytes associated with woody plants. Botryosphaeria and Neofusicoccum are two well known genera of the family. In this study 125 isolates morphologically resembling members of this family were collected from about 20 different fruit and forest trees in Iran. Based on morphology, MSP-PCR profile and DNA sequence data (ITS and tef1-a), four species were identified. Of these, Botryosphaeria dothidea, Neofusicoccum mediterraneum and N. parvum are known while Botryosphaeria scharifii is described here as new. N. mediterraneum is a new record for Iran and is reported here for the first time on mango trees. High diversity within Iranian population of N. parvum suggests the need to revise and reassess the morphological species description of N. parvum and closely related species.