Biochemical, pathogenical, antigenical, and molecular characteristics of Vibrio alginolyticus isolates from diseased cultured gilthead sea bream juveniles (4 and 20 g) were determined by laboratory challenge experiments. Intraperitonal challenge resulted in development of the disease but immersion did not. Electrophoresis of outer membrane protein (OMP) and lipopolysaccharides (LPS) was performed using sodium dodecyl sulphate poly-acrylamide gel electrophoresis (SIDS-PAGE). Molecular typing of Vibrio isolates was conducted using random amplification of polymorphic DNA (RAPD). According to SDS-PAGE of surface antigens of the isolates, profiles of both OMP and LIPS were heterogeneous. According to RAPID, isolated bacteria varied genetically. The findings reveal differences among V. alginolyticus isolates from diseased cultured gilthead sea bream in Turkey.