Technical Abstract: Vibrio scophthalmi has been considered as an opportunistic pathogen of the flat fish. There is little information available on V. scophthalmi adhesion to the host, an important step in the initial infection process. The objectives of this study were to (1) develop a modified Microbial Adherence to Hydrocarbons (MATH) assay using Colony Forming Unit (CFU) method and evaluate the cell surface hydrophobicity of V. scophthalmi, (2) detect slime layer and biofilm production of V. scophthalmi and (3) investigate the motility and antibiotic susceptibility associated with biofilm production of V. scophthalmi. The results showed that cell surface hydrophobicity of V. scophthalmi was moderate by Salt Aggregation Test (SAT) and MATH assay. The fraction partitioned to the hydrocarbon phase (%Adh) was determined significantly higher by modified CFU MATH assay than that by classical spectrophotometer MATH assay and microscope MATH assay. V. scophthalmi produced slime layer and biofilm. The optimal biofilm production conditions for V. scophthalmi were: BHIB media or TSB supplemented with 2% NaCl and 0-0.5% glucose and incubation for 24 h. The level of biofilm production is related with the pathogenicity of V. scophthalmi and antibiotics susceptibility. V. scophthalmi was motile with flagellum-mediated swimming and type IV pilus-mediated twitching, but no swarming.