Photoinactivation is a promising technique for Staphylococcus aureus control. This microorganism causes foodborne diseases (DTAs) and forms biofilms that are highly resistant and difficult to eradicate. Thus, the aim of this study was to evaluate the photodynamic activity of hypericin (HYP) in polymeric nanoparticles (Pluronic® P123) against S. aureus planktonic and biofilm cells. Planktonic cells and biofilms of S. aureus (ATCC 25923) were subjected to photoinactivation using low-power orange LED (0.3 mW/cm²) with different HYP formulation concentrations in Pluronic® P123. The P123 molar ratios were 2.5 (HYP/P123-2.5) and 10 (HYP/P123-10), respectively. The treatment times for planktonic cells were proposed by a mixture design, and bacterial photoinactivation was observed in concentrations of 12.5 to 3.12 μmol/L for HYP/P123-2.5 and reductions of ∼ 4.0 log CFU/mL in 12.5 to 0.78 μmol/L for HYP/P123-10. For biofilms, 30 min of darkness and 30 min of illumination were used. Maximum reductions were similar for both formulations and corresponded to approximately 0.9 log CFU/cm². It was concluded that photoinactivation with longer lighting times was effective against planktonic cells and could be potentially applied to control S. aureus.