Species in the family Botryosphaeriaceae are common pathogens causing fruit rot and dieback of many woody plants. In this study, 150 Botryosphaeriaceae isolates were collected from olive and other hosts in Spain and California. Representative isolates of each type were characterized based on morphological features and comparisons of DNA sequence data of three regions: internal transcribed spacer 5.8S, β-tubulin, and elongation factor. Three main species were identified as Neofusicoccum mediterraneum, causing dieback of branches of olive and pistachio; Diplodia seriata, causing decay of ripe fruit and dieback of olive branches; and Botryosphaeria dothidea, causing dalmatian disease on unripe olive fruit in Spain. Moreover, the sexual stage of this last species was also found attacking olive branches in California. In pathogenicity tests using unripe fruit and branches of olive, D. seriata isolates were the least aggressive on the fruit and branches while N. mediterraneum isolates were the most aggressive on both tissues. Isolates of B. dothidea which cause dalmatian disease on fruit were not pathogenic on branches and only weakly aggressive on fruit. These results, together with the close association between the presence of dalmatian disease symptoms and the wound created by the olive fly (Bactrocera oleae), suggest that the fly is essential for the initiation of the disease on fruit. Isolates recovered from dalmatian disease symptoms had an optimum of 26°C for mycelial growth and 30°C for conidial germination, suggesting that the pathogen is well adapted to high summer temperatures. In contrast, the range of water activity in the medium for growth of dalmatian isolates was 0.93 to 1 MPa, which was similar to that for the majority of fungi. This study resolved long-standing questions of identity and pathogenicity of species within the family Botryosphaeriaceae attacking olive trees in Spain and California.