Anthracnose caused by Colletotrichum spp. is the most damaging olive fruit disease in many countries, including Italy. This disease has been sporadically detected in Sicily, but new agronomic practices can increase risk of olive anthracnose in this region. An etiological study of the disease focused on local olive cultivars growing at the International Olive Germplasm Collection (IOGC) in Villa Zagaria, Enna, Sicily has been undertaken. During 2018 and 2019, 137 Colletotrichum strains were isolated from olives. Colony morphology, conidium characteristics, and multilocus sequence analyses aided identification of three species: C. acutatum (affecting 70% of symptomatic olives), C. gloeosporioides, and C. cigarro. Three C. acutatum strains (B13-16, P77, and P185), and one strain of each C. gloeosporioides (C2.1) and C. cigarro (Perg6B) were evaluated for pathogenicity on olive fruits from 11 Sicilian cultivars, known for their high-quality oil. Differences in virulence were detected among strains and their pathogenicity to the cultivars. The C. acutatum isolates were more virulent than those of C. gloeosporioides or C. cigarro. The Sicilian olive cultivars Cavaliera, Carolea, Calatina, and Nocellara del Belice were the most susceptible to the pathogen, while the cultivars Biancolilla and Nocellara Etnea were the most tolerant. Cultivar response under field conditions showed that anthracnose severity and fruit-rot incidence were positively correlated. This is the first report of C. acutatum and C. cigarro affecting olive trees in Sicily. Control measures for anthracnose depend on accurate characterization of the etiological agents and host cultivar resistance.