Nile tilapia is the most farmed freshwater fish species across vast areas of the African continent. Although Egypt is considered the largest African producer of Nile tilapia, this fish has been faced with frequent episodes of mortality at both hatchery and farm levels. In the present study, we conducted field screenings to identify the major bacterial pathogens responsible for these mass kills among broodstocks and seeds in Egyptian hatcheries. Aeromonas hydrophila and Streptococcus agalactiae were determined to be the major bacterial threats to hatchery-reared Nile tilapia. The bacterial isolates were presumptively identified using conventional biochemical tests and the API 20 NE miniaturized test. The final identities of the retrieved bacterial isolates were molecularly confirmed using PCR and sequencing of 16S rRNA genes. A. hydrophila and S. agalactiae isolates were found to be sensitive to florfenicol, while the two isolates exhibited resistance to novobiocin and ampicillin. The A. hydrophila isolates were confirmed to be sensitive to oxytetracycline, whereas the S. agalactiae isolates were sensitive to erythromycin. Histopathological examination of the livers of infected fish revealed vacuolar degeneration and necrosis of hepatocytes. Remarkably, infiltrations of Gram-positive cocci were documented within hepatic parenchyma and brain tissues. Further, the infected fish exhibited edematous brains with inflammatory cell infiltration through the meninges. Severe retinal pathology, including collagen fibre disorganization, oedema, and inflammatory cell infiltration was also detected. The current study emphasizes the deleterious impacts of some ubiquitous bacterial pathogens on the health status of Nile tilapia broodstocks and their seeds. Ultimately, we affirm that regular monitoring of water quality, feed quality, proper handling of broodstocks, and accurate diagnosis is a crucial asset in preventing disease spread and mass fish kills in Egyptian hatcheries.