The cost of Eucalyptus seed and adoption of strict phytosanitary regulations in seed trade makes it imperative to monitor the quality and ensure supply of pathogen-free seeds for both forestry regeneration and research. Based on seed health tests, two species in the Colletotrichum gloeosporioides species complex were found to be naturally associated with seeds of Eucalyptus dunnii, E. nitens and E. macarthurii produced in South Africa. Multiloci phylogenetic analyses based on the concatenated sequences of the ITS regions, β-tubulin, actin, glutamine synthetase and glyceraldehyde-3-phosphate dehydrogenase genes, identified representative isolates PPRI 24,314 as C. fructicola and PPRI 24,315 as C. kahawae subsp. cigarro (C. cigarro). Subsequent biochemical tests showed that isolate PPRI 24,315 was able to utilise either ammonium tartrate or citric acid as a sole carbon source, confirming its identity as C. cigarro. Pathogenicity tests showed that both C. fructicola and C. cigarro caused anthracnose leaf spots on E. camaldulensis, E. dunnii, E. nitens and E. viminalis seedlings. Disease symptoms included irregular dark-brown leaf spots on seedlings six days after inoculation. The two fungi were exclusively re-isolated from disease spots, thereby fulfilling Koch’s postulates. Sowing Eucalyptus spp. seed artificially inoculated with either of the two pathogens showed the seed-transmissibility of C. fructicola and C. cigarro in E. camaldulensis, E. dunnii and E. nitens seedlings. To our knowledge, this is the first report of C. fructicola and C. cigarro associated with Eucalyptus.