The aim of this study was to determine the frequencies of virulence genes and biofilm formation among Staphylococcus aureus clinical isolates, associated with lower respiratory infection. Different methods, from December 2007 to September 2009, were used to determine a total of 119 S. aureus clinical isolates, which were isolated from the sputum specimens of the patients with lower respiratory infections, verified by X-ray at a teaching hospital in Wenzhou, China. The present study has determined the frequencies of 9 virulence genes, including five classical major enterotoxin genes (sea to see), tst, eta, etb and lukS/F among these S. aureus isolates. Three multiplex PCRs and a single PCR were used for detecting the 9 virulence genes above. Microtiter-plate test was performed to semi-quantify biofilm formation of S. aureus. Results of all 9 tested virulence genes except see and etb, were detected among tested S. aureus isolates. All of 119 tested isolates, 79 isolates (68.1%) harbored at least one of the tested virulence genes, among which 76 isolates harbored only one gene tested and 3 isolates harbored two genes. The most prevalent virulence gene was sea (57.1%, 68/119). Only two isolates (1.7%) were positive for pvl. The prevalence of sea was significantly higher than those of other virulence genes (p < 0.01). The prevalence (62.8%) of sea among MRSA isolates was significantly higher than that (36.0%) among MSSA isolates (p < 0.01). Eighty-four (70.6%) of 119 isolates were biofilm producers. The prevalence of biofilm formation among MSSA isolates was significantly higher than that among MRSA isolates (p < 0.01). Conclusion, sea was the most prevalent virulence gene among the S. aureus clinical isolates associated with lower respiratory infection. Most of these isolates could form biofilm.