Thirty-nine Colletotrichum species have been reported as having all or part of their life cycle as pathogenic or endophytic to citrus globally, predominantly infecting leaves and twigs of multiple Citrus species. However, previous pathogenicity bioassays of Colletotrichum species on Citrus have mostly focused on wounding host tissues prior to inoculation, which leads to a biased measure of pathogenicity. Isolates of seven Colletotrichum species isolated from citrus in Australia were inoculated onto detached asymptomatic leaves, untreated with chemicals, 3 weeks after bud appearance, of lemon (Citrus × limon var. ‘Eureka’), Meyer lemon (Citrus × meyeri) and orange (Citrus sinensis var. ‘Washington Navel’) using a non-wound inoculation method. Colletotrichum australianum, C. gloeosporioides and C. siamense were the most aggressive species on Citrus species. Variable levels of aggressiveness were identified between isolates within C. australianum, C. fructicola, C. gloeosporioides, C. novae-zelandiae and C. siamense on certain Citrus species, suggesting multiple isolates of one Colletotrichum species need to be used in testing the disease severity of the pathogen on a host species. Colletotrichum isolates exhibited variation in aggressiveness among the three Citrus species. Lemon was relatively more resistant to Colletotrichum species. The grouping of disease severity was almost constant for all Colletotrichum isolates three and five days after inoculation. The development of a robust leaf-based pathogenicity bioassay has provided a better understanding of the pathogenicity of Colletotrichum species on Citrus, which will assist in the development of effective control strategies.