Tenacibaculum discolor develops biofilm in marine aquaculture production tanks and is identified as one of the causative agents of tenacibaculosis, a bacterial disease that causes significant losses in marine aquaculture production. In this study, the biofilm characteristics of T. discolor strain FMCC B487 were evaluated. Cell growth and biofilm formation and development were studied in miniaturized assays to assess the effect of different levels of environmental factors temperature and salinity, as well as the presence of monosaccharides potentially found in aquaculture hatcheries. The ability of the strain to grow and develop strong biofilms in ambient to high temperatures and at salinities above 20 g/L was shown. Mannose was the monosaccharide with the most prominent impact on the T. discolor strain FMCC B487 biofilm. The composition of planktonic cell extract, biofilm extracts, and extracellular polymeric substances (EPS) produced by T. discolor strain FMCC B487 were investigated by means of colorimetric and fluorometric assays as well as analyses by electrophoresis, gas chromatography, and high-performance size-exclusion chromatography coupled with a multiangle light scattering detector, revealing the dominance of proteins and lipids and the absence of high-molecular-weight polysaccharides. This information may serve as a basis for considering anti-biofilm strategies against the pathogen T. discolor.