Staphylococcus aureus (S. aureus) is commonly associated with hospital-acquired infections and is known to form biofilms. Bacteria inside biofilms display an increased resistance to chemotherapeutics and host immune defences. Efficient antibiotics or combination therapy are urgently needed to treat patients with biofilm-associated MRSA infections. The objective of the current study was to evaluate the in vitro antimicrobial activities of totarol alone or in combination with berberine chloride (BBR) against S. aureus grown in planktonic and biofilm cultures. The synergistic antimicrobial effects between BBR and totarol were observed in all tested strains grown in biofilms using a chequerboard microdilution method, with the fractional inhibitory concentration index values ranging from 0.125 to 0.375. No antagonistic activity was observed in any of the strains tested in suspension or biofilm cultures. The synergistic activity against S. aureus biofilms was also corroborated by confocal laser scanning microscopy and adhesion assays. Moreover, the present study demonstrated that combination BBR and totarol treatment effectively decreased the formation of S. aureus biofilms by affecting extracellular genomic DNA release and polysaccharide intercellular adhesin expression. Subsequently, real-time reverse transcriptase PCR analysis revealed that the combination of BBR and totarol effectively inhibited the transcription of the biofilm-related genes sarA, cidA and icaA. These results suggest that the combination of totarol and BBR is momentous for the further development of a therapy protocol against S. aureus biofilms.