This study was conducted to isolate and identify Aeromonas hydrophila which is the causative agent of motile Aeromonas septicemia MAS in freshwater fish (Oreochromis niloticus) and to study the efficacy and characterization of an antibacterial agent from Zingber officinaleagainst Aeromonas hydrophila in vitro which is causative agent of  MAS. This study was carried out using 100 O. niloticus weighting 80 ± 5 g Oreochromis niloticus were suffering from lose of appetite, lethargic, swim near the water surface, hemorrhage at the fins bases ulcer behind the head , roughness of scales (bristle out), slight distended abdomen and redness of anal opening. Internally the fish exhibited congestion of gills, liver, spleen, kidney and hemorrhage in peritoneal cavity, distended gall bladder, enlarged kidney and accumulation of serious fluid in abdominal cavity. The bacteriological examination of the samples taken from clinically affected fish  revealed the isolation of Aeromonas hydrophila . The incidence of infection in  O. niloticus reach 58% . The experimental infection with the isolated Aeromonas hydrophila intraperitoneally resulted in 90% mortality in O. niloticus  . The antibiogram sensitivity test revealed that ciprofloxacin in a dose of 5mg was highly effective in control of Aeromonas hydrophila. The separation of the active ingredient and its purification was performed using both thin layer (TLC) and column chromatography techniques. The physico-chemical characteristics of the purified antibacterial agent viz. color, melting point, solubility, elemental analysis, spectroscopic characteristics and assay of total phenolics have been investigated. An imperical formula ofZingber officinale a suggested empirical formula of C11 H19 O8N which was evaluated as an antibacterial agent against A. hydrophila organism.