Eucalyptus species are among the most important forest crops in the world and can be affected by several pathogens, mainly by fungi of the genus Pestalotiopsis, which cause leaf spots. Studies aimed at the pathogenic characterization of Pestalotiopsis spp. from lesions of eucalyptus leaves in Brazil are still limited. The objective of this work was to evaluate the pathogenic potential of Pestalotiopsis grandis-urophylla isolates. For that, healthy leaves of adult Eucalyptus grandis ‘GG 100’ plants were inoculated with mycelial suspension of different P. grandis-urophylla isolates. The leaves inoculated with the pathogen were submitted to controlled conditions in a humid chamber in transparent acrylic gerbox boxes. Disease severity assessments were performed at 4, 6, 8 and 10 days after inoculation. Isolate E 72-04 had the highest area under the disease progress curve (AUDPC). Regarding the development of lesions, isolates E-72-02 and E-72-03 fit the polynomial model of the second degree, while isolate E-72-04 fit a linear model. The methodology tested reproduced typical symptoms of Pestalotiopsis and can be used as a parameter for new pathogenicity tests with this fungal genus.