Cladode brown spot (CBS) is an important disease of Nopalea cochenillifera in the semiarid region of Northeastern Brazil. It has been reported in several countries, but its etiology is controversial, attributed to a complex of pathogens. Fifty fungal isolates were obtained and identified based on morphology and phylogeny, through analysis of the ribosomal DNA internal transcribed spacers (ITS), translation elongation factor 1-alpha (TEF1-alpha), β-tubulin (TUB-2), second largest subunit of RNA polymerase (RPB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the mating type locus MAT1–2 (ApMAT) partial gene sequences. Seven fungal general and twenty species were identified – Alternaria sp., A. longipes and A. tenuissima; Colletotrichum fructicola and C. siamense; Fusarium lunatum, F. incarnatum, and F. verticillioides; Lasiodiplodia euphorbicola, L. iraniensis, L. pseutheobromae, L. theobromae and Lasiodiplodia sp.; Neofusicoccum batangarum; Neopestalotiopsis australis, N. protearum and Neopestalotiopsis sp.; and Nigrospora sphaerica, N. hainanensis and Nigrospora sp. Four of these species had already been reported on Cactaceae of the genus Opuntia, but only N. batangarum, C. fructicola and C. siamense have been reported to date on the genus Nopalea. All isolates were pathogenic to detached cladodes of N. cochenillifera ‘Miúda’. Differences in aggressiveness were observed among the species, with L. iraniensis and F. lunatum characterized as the most aggressive species, whilst F. verticillioides as the least aggressive. This study provide important information on the fungi associated with cladode brown spot and improve the strategies for the management of disease on N. cochenillifera.