The interest in production of buffalo milk is increasing in Brazil due to its physico-chemical and nutritional characteristics. However, just as sheep, goats and cows are susceptible to mastitis, so are buffaloes, which is mainly caused by Staphylococcus aureus. In this regard, biofilm formation, the ability to escape host immune defense and virulence factor, is presumably a key factor for acute and chronic intra-mammary infection in buffaloes. In this study, biofilm forming capabilities and virulence genes were evaluated in S. aureus isolated from buffalo milk and milking environments, using phenotypic and genotypic assays. Thirty two S. aureus strains isolated from buffalo milk, milking machines and milkers’ hands were obtained from a farm in Analândia, São Paulo State, Brazil. Samples were collected in April, June, October and November 2013. These strains were tested for the presence of sa442, icaA, icaD, clfA, clfB, sarA and hla genes, slime production using Congo Red Agar (CRA), and biofilm formation using microtiter assay. All samples of S. aureus were positive for CRA and microtiter assay. Although, icaA and icaD genes were simultaneously detected in nine of the 32 samples, none of the samples were positive for icaA; only seven were positive for icaD gene. This suggests that other factors may be involved in biofilm formation. Seventeen strains of S. aureus were positive for sarA gene, nine for clfA gene, 16 for clfB and 16 for hla. A great variability in SmaI restriction profiles of S. aureus strains was observed. Thirty isolates were typified and two strains were not by SmaI restriction. In addition, 18 pulse types were detected. It is hypothesized that biofilm can be produced by the expression of icaD gene only. Our findings suggest that S. aureus strains from buffalo milk and milking environment are similar, which contradict the findings obtained from bovine strains. This behavior may contribute to the persistence of mastitis in buffalo caused by S. aureus, which results in a potential zoonotic problem. Our results may bring new insights into the development of novel strategies for the prevention and treatment of bubaline mastitis.