Species of the Botryosphaeriaceae are important pathogens causing cankers and die-back on many woody plants. In Uruguay, Neofusicoccum eucalyptorum, N. ribis and B. dothidea have previously been associated with stem cankers on plantation-grown Eucalyptus globulus. However, very little is known regarding the occurrence and species diversity of Botryosphaeriaceae in native Myrtaceae forests or what their relationship is to those species infecting Eucalyptus in plantations. The objectives of this study were to identify the Botryosphaeriaceae species present as endophytes or associated with cankers in both introduced and native tree hosts in Uruguay, and to test the pathogenicity of selected isolates obtained from native trees on Eucalyptus. Symptomatic and asymptomatic material was collected countrywide from Eucalyptus plantations and native Myrtaceae trees. Single spore cultures were identified based on conidial morphology and comparisons of DNA sequences of the ITS and EF1-α regions. Six Botryosphaeriaceae species were identified. Botryosphaeria dothidea, N. eucalyptorum and specimens residing in the N. parvum-N. ribis complex were isolated from both introduced Eucalyptus and native Myrtaceae trees, whereas Lasiodiplodia pseudotheobromae was found only on Myrcianthes pungens. Diplodia pseudoseriata sp. nov. and Spencermartinsia uruguayensis sp. nov. are novel species found only on native myrtaceous hosts. Pathogenicity tests showed that isolates obtained from native trees and identified as L. pseudotheobromae, N. eucalyptorum and the N. parvum-N.ribis complex are pathogenic to E. grandis. Interestingly, Lasiodiplodia pseudotheobromae has not previously been found on Eucalyptus in Uruguay and represents a potential threat to this host.