Eighteen different fungal species were isolated from symptomatic wood of olive trees (Olea europaea) affected by twig and branch dieback in California and identified by means of morphological characters and multigene sequence analyses of the internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2), a partial sequence of the β-tubulin gene, and part of the translation elongation factor 1-α gene (EF1-α). These species included Diaporthe viticola, Diatrype oregonensis, Diatrype stigma, Diplodia mutila, Dothiorella iberica, Lasiodiplodia theobromae, Phaeomoniella chlamydospora, Phomopsis sp. group 1, Phomopsis sp. group 2, and Schizophyllum commune, which are for the first time reported to occur in olive trees; Eutypa lata, Neofusicoccum luteum, Neofusicoccum vitifusiforme, and Phaeoacremonium aleophilum, which are for the first time reported to occur in olive trees in the United States; and Botryosphaeria dothidea, Diplodia seriata, Neofusicoccum mediterraneum, and Trametes versicolor, which have been previously reported in olive trees in California. Pathogenicity studies conducted in olive cultivars Manzanillo and Sevillano showed N. mediterraneum and Diplodia mutila to be the most virulent species and Diatrype stigma and D. oregonensis the least virulent when inoculated in olive branches. Intermediate virulence was shown for the rest of the taxa. This study demystifies the cause of olive twig and branch dieback and elucidates most of the fungal pathogens responsible for this disease in California.