The dramatic increase of bacteria resistant to antibiotics makes it unlikely that the discovery of new classical drugs will solve this problem permanently. Therefore, new antimicrobial approaches are needed to treat antibiotic-resistant Staphylococcus aureus infections. Photodynamic inactivation (PDI) emerges as a photochemotherapeutic approach with applications in antimicrobial therapy. The PDI process is based on the combined use of light, oxygen, and a photosensitizing (PS) agent. These three components interact to generate reactive oxygen species, which are cytotoxic and irreversibly damage the vital components of microbial cells, causing death. In this work, the photoantimicrobial effect of zinc(II)2,9,16,23-tetrakis[4-(N-methylpyridyloxy)]phthalocyanine (ZnPPc⁴⁺) on different S. aureus strains was evaluated, comparing its action on several virulence factors (including hemolysin, lipase and lecithinase activity, mannitol fermentation, catalase production and carotenoid content) before and after photodynamic treatment. Finally, the ability to produce biofilms pre- and post-treatment was analyzed. The results indicate that ZnPPc⁴⁺ is an effective PS to photoinactivate different strains of planktonic S. aureus, at low concentration and light doses, and to significantly reduce the number of microorganisms that are part of biofilm. Furthermore, ZnPPc⁴⁺ was shown to alter the expression of some virulence factors tested. Therefore, PDI may become a promising alternative therapy, not only to control the reproduction of pathogenic microorganisms, but also to reduce the effects of virulence factors that may remain after the inactivation of microorganisms.