The present study investigates the effect of surface hydrophobicity and media on in vitro biofilm formation potential of S. aureus isolates from two dairies. There was lack of correlation between biofilm formation potential of S. aureus and sampling points but not with the source of isolation. Biofilm formation was more pronounced on hydrophobic polypropylene (PP) than hydrophilic stainless steel (SSC). A better correlation was established between the biofilms on SSC and PP when analyzed by crystal violet staining than conventional plating method. On comparative evaluation of nutrient rich and diluted growth media, enhanced biofilm formation was observed in latter (1/20 tryptic soya broth). Highest survival in the preformed biofilms was observed at incubation temperature of 25 °C in sterilized skim milk rather than TSB. The present study signify preferable adherence of S. aureus on hydrophobic surfaces and potential survival within preformed biofilm at temperatures prevalent in dairy industries.