Photodynamic antimicrobial chemotherapy (PAC) is an efficient tool for inactivating microorganisms. This technique is a good approach to inactivate the foodborne microorganisms, which are responsible for one of the major public health concerns worldwide-the foodborne diseases. In this work, response surface methodology (RSM) was used to evaluate the interaction of Eosin Y (EOS) concentration and irradiation time on Staphylococcus aureus counts and a sequence of designed experiments to model the combined effect of each factor on the response. A second-order polynomial empirical model was developed to describe the relationship between EOS concentration and irradiation time. The results showed that the derived model could predict the combined influences of these factors on S. aureus counts. The agreement between predictions and experimental observations (R2adj = 0.9159, p = 0.000034) was also observed. The significant terms in the model were the linear negative effect of photosensitizer (PS) concentration, followed by the linear negative effect of irradiation time, and the quadratic negative effect of PS concentration. The highest reductions in S. aureus counts were observed when applying a light dose of 9.98 J/cm2 (498 nM of EOS and 10 min. irradiation). The ability of the evaluated model to predict the photoinactivation of S. aureus was successfully validated. Therefore, the use of RSM combined with PAC is a promising approach to inactivate foodborne pathogens.