This study aimed to investigate the in silico biofilm production ability of Staphylococcus aureus strains isolated from milking parlor environments on dairy farms from São Paulo, Brazil. The Staph. aureus isolates were obtained from 849 samples collected on dairy farms, as follows: milk from individual cows with subclinical mastitis or history of the disease (n=220); milk from bulk tank (n=120); surfaces of milking machines and utensils (n=389); and milk handlers (n=120). Thirty-one Staph. aureus isolates were obtained and categorized as pulsotypes by pulsed-field gel electrophoresis and submitted to assays for biofilm formation on polystyrene, stainless steel, rubber, and silicone surfaces. Fourteen (45.2%) pulsotypes were considered producers of biofilm on the polystyrene microplate assay, whereas 13 (41.9%) and 12 (38.7%) pulsotypes were biofilm producers on stainless steel and rubber, respectively. None of the pulsotypes evaluated produced biofilms on silicone. Approximately 45% of Staph. aureus pulsotypes isolated from different sources on dairy farms showed the ability to produce biofilms in at least one assay, indicating possible persistence of this pathogen in the milking environment. The potential involvement of Staph. aureus in subclinical mastitis cases and its occurrence in milk for human consumption emphasize the need to improve hygiene practices to prevent biofilm formation on the farms studied.