BACKGROUND: Mastitis is an important disease that affects dairy herds worldwide. The Staphylococcus aureus (S. aureus) is the causative pathogen for mastitis. This pathogen has the tendency to biofilm forming, and may happen to antibiotic resistance. OBJECTIVES: The aim of this study was to characterize the biofilm formation of different genotypes and antibiotic resistance pattern of S. aureus isolated from the subclinical bovine mastitis in Tehran province. METHODS: The lactating dairy cows were screened for the subclinical mastitis. The isolates were identified by phenotypic method and the presence of the nuc gene. The biofilm forming and quantification was characterized using colorimetric assay. The S. aureus biofilm gene was evaluated using PCR assay. The antimicrobial susceptibility of the isolates was assessed using DAD method. The lowest antimicrobial concentration preventing the visible growth was construed by MIC50. The antibiotic susceptibility and MBECs for the bacteria embedded in the biofilms were determined by XTT method. RESULTS: The antimicrobials susceptibility test showed penicillin and ceftiofur to be less and more effective in vitro, respec-tively. The genotypic characterization showed that the highest and the lowest frequencies for icaD (75%) and fnbB (31.2%) genes, respectively. The biofilm formation was also characterized. The MBEC results for the bacterial biofilm showed resistance to ceftiofur in the biofilm state; however, these strains were susceptible to this agent in the planktonic state. CONCLUSIONS: The biofilm formation is a significant virulence factor that was detected at a high rate. It is antibiotic-resistant and responsible for the subclinical bovine mastitis that does not respond to the routine treatments.In order to control the infection achieve the effective treatment, and prevent the emergence of antibiotic-resistant bacteria, it is necessary to isolate the causative agent and determine the antimicrobial susceptibility