<p>Background: Biofilm infections are a major challenge in medical practice. Bacteria that live in a biofilm phenotype are more resistant to both antimicrobial therapy and host immune responses compared to their planktonic counterparts. So, there is need for new therapeutic strategies to combat these infections. A promising approach [known as Photodynamic Inactivation (PDI)] to kill bacteria growing as biofilms uses light in combination with a photosensitizer to induce a phototoxic reaction which produces reactive oxygen species that can destroy lipids and proteins causing cell death. PDI does not always guarantee full success, so, combination of PDI with antibiotics may give increased efficiency. This study aimed to determine if PDI was effective in the eradication of <em>Staphylococcus aureus (S. aureus)</em> biofilms in combination with linezolid.<br /> Methods: The susceptibility of biofilm cultures of three <em>S. aureus</em> strains to Methylene Blue (MB) and Toluidine Blue O (TBO)-mediated PDI was determined alone and in combination with linezolid.<br /> Results: Bactericidal activity (&ge;3 log₁₀ reduction in viable cell count) was not achieved with MB/TBO-PDI or antibiotic treatment alone. When antibiotic treatment was combined with TBO-PDI, a greater reduction in viable count than antibiotic alone was observed for two strains.<br /> Conclusion: This study showed that although TBO-PDI did not have good bactericidal activity against <em>S. aureus</em> biofilms; it increased the antimicrobial activity of linezolid against these bacteria.</p>