Local bacterial cultures could be isolated from 200 Oreochromis niloticus and were caught as random samples from ELAbassa, Abou- Hammad, and Sharkia, Egypt. Fish farms with an average body weight 40±5g. Suffered From signs of septicemia as hemorrhages on several parts of the body surface (mouth) base of fins, abdominal part. opercula , and around the anal opening  turbidity of the eyes and slight exophthalmia roughness of scales and sometime scale losses, postmortemally showing, hepatomegaly, splenomegaly, congestion of gills, kidney and accumulation, of bloody fluid in abdominal wall to bacterial strains are Gram negative bacteria were isolated from fish. The bacterial isolate taxonomic classification clarified that the bacterial isolates was likely belonging to Pseudomonas aeruginiosa-I, Aeromonas hydrophila-2 according to its physiological morphological, and biochemical characters. The active extract using bioactivity-guided technique of aqueous and organic extracts of (Eugenia caryophllate). The separation of the active ingredient and its purification was performed using both thin layer chromatography (TLC) and column chromatography techniques. The physico-chemical characteristics of the purified antibacterial agent viz. color, melting point, solubility, elemental analysis and spectroscopic characteristics (GC – mass techniques) have been investigated. This analysis indicates a suggested empirical formula of C₁₀ H₁₂ O₂. The biological activities i.e. MICs of the Purified antibacterial agent were also determined.