Abstract Olive production has expanded strongly in the last twenty years in Uruguay. In this study, a collection of 37 Botryosphaeriaceae isolates obtained from stem canker and fruit rot symptoms in olive trees of six cultivars from 14 orchards throughout the country were characterized. Based on phylogenetic analysis of ITS and TEF‐1α gene regions and phenotypical characteristics, eight species belonging to the genera Botryosphaeria (41%), Neofusicoccum (51%) and less frequently to Diplodia (8%) were identified. The recognized species were B . wangensis ( n = 11), N . cryptoaustrale ( n = 9), N . luteum ( n = 7), B . dothidea ( n = 4), N . occulatum ( n = 2), D . seriata ( n = 2), N . australe ( n = 1) and D . mutila ( n = 1). Isolates of all Botryosphaeriaceae species were able to cause typical necrotic lesions on inoculated olive stems of Arbequina cultivar under laboratory conditions, but those belonging to Neofusicoccum genus were significantly more aggressive. Five of the eight species found in this study were previously reported affecting other fruit and forestry trees in Uruguay; thus, further epidemiologic studies and management programmes should be developed considering all the Botryosphaeriaceae hosts, especially those cultivated in proximity. Also, we present B . wangensis , N . cryptoaustale , N . occulatum and D . mutila species as new records to olive worldwide.