Botryosphaeriaceae are common endophytes of trees. Some species are also known to be pathogens. It is, therefore, assumed that endophytic Botryosphaeriaceae are often involved in general die-back diseases. Here we test this assumption in severe branch die-back observed on Acacia karroo trees in the Pretoria area of South Africa. The presence of the Botryosphaeriaceae was compared between healthy and diseased tissue on the same trees. Eight Botryosphaeriaceae species were isolated from die-back and healthy branches. Of these, six species, namely Tiarosporella urbis-rosarum, Diplodia allocellula, Phaeobotryosphaeria variabilis, Dothiorella brevicollis and Neofusicoccum vitifusiforme were obtained from healthy tissues, and only two species, Dothiorella dulcispinae and Spencermartinsia pretoriensis, were exclusively found in die-back branches. Spencermartinsia viticola was found in both tissue types and this fungus was also the most commonly isolated species from both healthy and die-back samples. Results of pathogenicity trials showed highly variable results for the isolated species and that the two species associated only with die-back symptoms, were weakly pathogenic. These results suggest that the Botryosphaeriaceae found in these trees were not directly associated with the die-back symptoms, despite their diversity and common occurrence in these tissues. The situation is different in other tree systems where dominant species, often with wide host ranges, have been shown to be involved in die-back diseases. This indicates the importance of characterizing the unique aspects of each tree disease system.