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Background & aim: Regarding to the importance of S. aureus colonization in bovine mastitis and the role of biofilm in its pathogenesis, the aim of this study was to determine the phenotypic production of biofilm and subsequently identify genes related to biofilm production in isolates of S. aureus from bovine with subclinical mastitis. Methods:In the present cross-sectional study, a total of 502 milk samples were collected from bovine with subclinical mastitis in Boyerahmad and Dena townships. After isolation of bacteria was completed, the detection of nucA gene by PCR method was conducted for the final confirmation of S. aureus. Congo Red Agar plate was used for the assessment of biofilm production. The PCR method was used for the detection of icaA, icaD, fnbA, clfA, cna and bap genes. The collected data was analyzed by the SPSS software version 15 and chi-square test.Results: A total of 80 (15.9%) isolates of Staphylococcus aureus were isolated, of which 55 (68.7%) isolates were able to form biofilms. The highest frequency of icaD gene was identified in 87.5% of isolates and bap gene with the lowest frequency (5%). Significant association were observed between biofilm production and presence of icaD(p=0.0001), icaA(p=0.003), fnbA(p=0.0001) and clfA(p=0.0001). Conclusion:Considering the important role of biofilm in development of antibiotic resistance and high frequency of biofilm producer isolates, this finding should be considered as an alarm. Hence, the biofilm production helps the bacterial colonization and the pathogenesis could lead to economical and healthcare burden on the community.