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This work aimed to determine the presence of bacterial pathogens in fish with a clinical picture suggestive of infectious disease in Nile tilapia reared in Chiapas, Mexico. Blood and viscera samples were taken from healthy and diseased animals from commercial farms. Clinical and pathological examinations of each individual were performed and samples were collected for bacteriological studies. The bacterial isolates were identified and characterized by culture, biochemical tests, antibiogram, challenge tests and 16S rRNA sequencing. Staphylococcus haemolyticus and Providencia vermicola were isolated from various diseased organisms. The clinical picture caused by Staphylococcus haemolyticus was characterized by appetite disorders, neurological signs, nodulation or ulceration in different areas and congestion or enlargement of internal organs. Providenciosis in juvenile specimens caused a characteristic picture of hemorrhagic septicemia. Challenge tests performed in healthy organisms revealed that both infections caused higher mortality rates in fish (p < 0.05) compared with non-infected specimens, with 100% survival. There was 100% mortality for animals infected with P. vermicola after three days post infection and 45% for those infected with S. haemolyticus. The isolation and identification of two pathogens involved in an infection process were achieved and cataloged as potential causal agents of disease outbreaks in tilapia farming in Mexico. This is the first report of possible bacterial infection caused by S. haemolyticus and P. vermicola in tilapia farms, which are two uncommon but potentially emerging pathogens for the species.