Although the potent antibacterial ability of radezolid againstStaphylococcus aureushas been widely reported worldwide, its antibacterial and anti-biofilm activity against theS. aureusclinical isolates from China remains elusive. In this study, the minimum inhibitory concentration (MIC) of radezolid was determined inS. aureusclinical isolates from China using the agar dilution method, and the relationship between radezolid susceptibility and ST distribution was also investigated. The anti-biofilm activity of radezolid againstS. aureuswas determined by a crystal violet assay and compared with that of linezolid and contezolid. The quantitative proteomics ofS. aureustreated with radezolid was analyzed, and the genetic mutations in radezolid-induced resistantS. aureuswere determined by whole-genome sequencing. The dynamic changes in transcriptional expression levels of several biofilm-related genes were analyzed by quantitative RT-PCR. Our data showed that radezolid MIC ranged from ≤0.125 to 0.5 mg/L, which was almost 1/4 × MIC of linezolid againstS. aureus, indicating the greater antibacterial activity of radezolid than linezolid. TheS. aureusclinical isolates with radezolid MICs of 0.5 mg/L were most widely distributed in ST239 of MRSA and ST7 of MSSA. Moreover, the more robust anti-biofilm activity of radezolid with subinhibitory concentrations (1/8 × MIC and 1/16 × MIC) was demonstrated againstS. aureuswhen compared with that of contezolid and linezolid. Genetic mutations were found inglmS, 23S rRNA, and DUF1542 domain-containing protein in radezolid-induced resistantS. aureusselected byin vitroinduction of drug exposure. Quantitative proteomic analysis ofS. aureusindicated that the global expression of some biofilm-related and virulence-related proteins was downregulated. Quantitative RT-PCR further confirmed that the expressions of some downregulated biofilm-related proteins, includingsdrD, carA,sraP,hlgC,sasG,spa,sspP,fnbA, andoatA, were decreased after 12 h and 24 h of exposure to radezolid. Conclusively, radezolid shows robust antibacterial and anti-biofilm activity againstS. aureusclinical isolates from China when compared with contezolid and linezolid.