To evaluate the effectiveness of the photodynamic therapy using hypericin (HYP) against both planktonic and biofilm-forming Staphylococcus aureus. HYP photoactivity was evaluated against methicillin-susceptible and resistant S. aureus. Bacterial suspension or biofilm were preincubated with HYP and subjected to LED illumination. Viable bacteria were determined by colony counting. Preincubation with HYP (5 min) plus light exposure (10 min) showed bactericidal effect against planktonic methicillin-susceptible S. aureus and methicillin-resistant S. aureus. Longer preincubation times (24 h) and time light exposure (30 min) were required to reach HYP-photoactivity against S. aureus biofilms. HYP-photoactivity was correlated to the biofilm production. HYP could be a potential photosensitizer for the inactivation of staphylococcal biofilms forming on the surfaces accessible to visible light.