Staphylococcus aureus is a pathogenic bacterium capable of developing biofilms on food-processing surfaces, a pathway leading to cross contamination of foods. The purpose of this study was to investigate the influence of environmental stress factors found during seafood production on the adhesion and biofilm-forming properties of S. aureus. Adhesion and biofilm assays were performed on 26 S. aureus isolated from seafood and two S. aureus reference strains (ATCC 6538 and ATCC 43300). Cell surface properties were evaluated by affinity measurements to solvents in a partitioning test, while adhesion and biofilm assays were performed in polystyrene microplates under different stress conditions of temperature, osmolarity, and nutrient content. The expression of genes implicated in the regulation of biofilm formation (icaA, rbf and σ B ) was analyzed by reverse transcription and quantitative real time PCR. In general, S. aureus isolates showed moderate hydrophobic properties and a marked Lewis-base character. Initial adhesion to polystyrene was positively correlated with the ionic strength of the growth medium. Most of the strains had a higher biofilm production at 37 °C than at 25 °C, promoted by the addition of glucose, whereas NaCl and MgCl2 had a lower impact markedly affected by incubation temperatures. Principal Component Analysis revealed a considerable variability in adhesion and biofilm-forming properties between S. aureus isolates. Transcriptional analysis also indicated variations in gene expression between three characteristic isolates under different environmental conditions. These results suggested that the prevalence of S. aureus strains on food-processing surfaces is above all conditioned by the ability to adapt to the environmental stress conditions present during food production. These findings are relevant for food safety and may be of importance when choosing the safest environmental conditions and material during processing, packaging, and storage of seafood products.