Biofilm-associated infection due to Staphylococcus epidermidis, the leading nosocomial pathogen, is a major problem for the public health system, but the regulation of this important phenotype is not completely understood. Using a highly discriminatory screening procedure for genes that influence biofilm formation, we identified the transcriptional regulator SarZ as a novel important determinant of biofilm formation and biofilm-associated infection, on the basis of the significant impact of sarZ on the transcription of the biosynthetic operon for biofilm exopolysaccharide. In addition, sarZ influenced the expression of a series of virulence genes, including genes that influence the expression of lipases and proteases, resistance to an important human antimicrobial peptide, and hemolysis. Our study indicates that the SarZ regulator has a key role in maintaining the typical S. epidermidis phenotype, which is characterized by pronounced biofilm formation and immune evasion, a likely reason for the success of S. epidermidis as a colonizing organism and pathogen in chronic, biofilm-associated infection.