Brazilian aquaculture has shown a strong expansion. Such growth has been possible due to the large availability of freshwater, the increase in the fish consumption and the great productive potential presented by some native fish species, such as cacharas, Pseudoplatystoma fasciatum, and pintados, Pseudoplatystoma corruscans. However, concomitantly with the production, the occurrence of disease outbreaks associated to infectious and parasitic diseases, resulting in economic losses. Thus, the etiological diagnosis of these diseases is extremely important, leading to the development of treatments and, mainly, to the prevention of new outbreaks, collaborating in the implementation of effective sanitary measures. In the present study, a fish farmer of the interior of the state of São Paulo, went to the Lab. Higiene Zotécnica reporting a mortality of 90% of a lot of juvenile pintado. 20 juveniles of the lot were sent, showing erratic swimming in circles and anorexia. The fish were washed with 5% chlorin solution and, in sterile environment, the kidney, spleen and liver were dissected, macerated and homogenized in one milliliter of BHI broth, which was plated on blood agar at 37°C for 24hs. The growth colonies were subcultured to obtain isolates. Simultaneously, the extraction of nucleic acids (DNA and RNA) of the collected tissues was performed for family Iridoviridae and TiLV differential laboratory diagnosis by PCR. Additionally, the obtained isolates were submitted to Gram staining, catalase test, MALDI-TOF, PCR for 16S and RNA polymerase (RpoD) genes, nucleotide sequencing and antibiogram. All the samples were negative for the researched viruses. However, the isolated bacteria were positive for catalase and, according to Gram staining, it was possible to observe the presence of two types of Gram-negative bacilli of different sizes. According to the MALDI-TOF, the isolates belonged to the genera Aeromonas spp. and Plesiomonas spp. Nucleotide sequencing of PCR products for the RpoD gene and GenBank (Blastn) search for similarity confirmed two distinct species of bacteria in the fish analyzed: Aeromonas jandaei and Plesiomonas shigelloides. Phylogenetic reconstruction, using the neighbor-joining method based on sequences of the RpoD gene from different species of Aeromonas spp., grouped the Brazilian isolate into the A. jandaei clade with high associated bootstrap value (92). On antibiogram, the detected A. jandaei bacterium was resistant to Ampicillin, Cephalothin, Clindamycin, Gentamicin, Oxacillin, Norfloxacin and Penicillin G. The P. shigelloides isolate was resistant to Clindamycin, Cephalothin, Gentamicin, Norfloxacin, Oxacillin, Penicillin G, Sulfazotrim and Tetracycline. Mortality outbreaks caused by Aeromonas spp. in association with P. shigelloides is very common in aquaculture and can cause serious economic losses to fish farmers. In this context, as later stages of the present study, we intend to perform molecular and phenotypic characterization of the degree of virulence of isolates in in vitro (virulence genes) and in vivo (experimental infections) assays in order to better understand the pathogenicity of these agents for native fish species from Brazil, collaborating with subsidies for the implementation of more adequate sanitary management protocols in the country.