Aims: To identify bacterial pathogens of diseased NiIe tilapia Oreochromis niloticus and determine their virulence. Methods and Results: Sixteen bacterial isolates were recovered from diseased Nile tilapias (O.niloticus) reared in floating cages in Adolfo Lopez Mateos (ALM), Sanalona and Dique IV dams in Sinaloa, Mexico, from February to May 2009. The bacterial isolates were identified by phenotypic and molecular (rep-PCR and 16S rRNA sequencing) methods and were mostly isolated from the kidneys and the brain of tilapias. Bacterial cells and extracellular products (ECPs) of strains were characterized and used in experimental infections with sole Solea vulgaris and Mozambican tilapia Oreochromis mossambicus. The fish challenged with Aeromonas dhakensis sp. nov. comb nov, Pseudomonas mosselii and Microbacterium paraoxydans (3.1 x 10(6) CFU g(-)1) exhibited mortality between 40 and 100% starting at 6h postinoculation. The ECPs displayed gelatinase, haemolytic and cytotoxic activity, causing the total destruction of the HeLa cell lines. Conclusions: Aeromonas dhakensis and Ps.mosselii were virulent to O. mossambicus, whereas Mic.paraoxydans displayed virulence to S.vulgaris. Significance and Impact of the Study:This the first time that Aeromonas dhakensis and Ps.mosselii are reported as pathogens to tilapia and Mic.paraoxydans was isolated from fish; then, these fish pathogens could be a threat to farmed Nile tilapia in Mexico.