In the current study, clinically diseased cultured Oreochromis niloticus at various life stages were collected from different fish farms within the governorates of Qalyubia, Kafr El-Sheikh, Sharqia, and Port Said. The clinical pictures and gross lesions were recorded. Bacterial pathogen isolation and identification were accomplished using both traditional and molecular techniques. For molecular characterization, traditional PCR was employed to confirm the biochemically identified bacteria using the 16S rRNA. The pathogenicity of the isolates was examined, and histopathological findings were recorded for each. At the farm site examination, the infected fish displayed general septicemic signs such as skin hemorrhages and ulcerations, uni- and bilateral exophthalmia, congested internal organs, and significant mortality. The overall prevalence of bacterial infection was (26.2%). Streptococcus agalactiae was the most prevalent bacteria recovered from clinically diseased juveniles (15.5%), with the summer season exhibiting the highest incidence. The retrieved bacterial isolates were Streptococcus agalactiae (S. agalactiae) (50 isolates, 15.5%), Streptococcus faecalis (S. faecalis) (5 isolates, 1.5%), Enterococcus faecium (E. faecium) (37 isolates, 11.4%), and Lactococcus garviae (L. garviae) (55 isolates, 17%) were isolated from infected juveniles in the autumn (55 isolates, 17%) and adults in the summer (20 isolates, 6.2%). According to the results of this investigation, streptococcal infection, specifically S. agalactiae, S. faecalis, E. faecium, and L. garviae (strain I and II), could be a significant contributor to tilapia mortality during the summer.