This study was carried out to investigate the effect of florfenicol on Oreochromis niloticus experimentally infected with Enterococcus faecalis and evaluation of florfenicol residue in both infected and non-infected fish. Fish was divided into four equal groups. Group (I) was kept as control, group (II) was fed on florfenicol treated diet at dose (15 mg/kg B.W/day), group (III) was experimentally infected intramuscular (IM) with Enterococcus faecalis (1.2x108 CFU/ml) and fed on control diet. The last group (IV) was experimentally infected with Enterococcus faecalis (1.2x108 CFU/ml) then fed on florfenicol treated diet for 10 successive days. Serum samples were taken on 1st, 7th, 14th and 21st days post 10 days treatment for biochemical assay and evaluation of liver and kidney functions. The experimentally infected treated group with florfenicol revealed significant elevation (P≤ 0.05) in serum total protein, albumin, globulin, liver enzymes and kidney functions on 1st day post feeding in compared to other groups. These parameters return to normal with no significant difference on 7th, 14th and 21st days post treatment. Florfenicol residue was found to be higher in musculature than liver in infected treated group compared to non infected treated group on 1st day post feeding. This concentration decreased gradually in musculature and liver on 4th and 7th days post feeding. Histopathological examination of liver and kidneys of infected treated O. niloticus revealed moderate congestion and thickening in the wall of central vein and hypercellularity of some glormeuli compared to experimentally infected group with Enterococcus faecalis. In conclusion, florfenicol is considered the most effective approved antibiotic used for treatment of enterococcosis in Nile tilapia and its residue in musculature and liver reach to below the maximum recommended level (1µg/g) at 7th day post treatment.