This study investigated the efficacy of pulsed UV-light for continuous-flow milk treatment for the inactivation of Staphylococcus aureus, a pathogenic microorganism frequently associated with milk safety concerns. Pulsed UV light is an emerging technology, which can be used for the inactivation of this pathogen in milk in a relatively short time. Pulsed UV light damages the DNA of the bacteria by forming thymine dimers that lead to bacterial death. The effect of sample distance from the quartz window of the UV-light source, number of passes, and flow rate was investigated. A response surface methodology was used for the design and analysis of experiments. Milk was treated at 5-, 8-, or 11-cm distance from a UV-light strobe at 20, 30, or 40 mL/min flow rate and treated up to 3 times by recirculation of milk to assess the effect of the number of passes on inactivation efficiency. Log10 reductions varied from 0.55- to 7.26-log10 CFU/mL. Complete inactivation was obtained in 2 cases and no growth was observed following an enrichment protocol. Predicted results were in agreement with the experimental data. Overall, this work demonstrates that pulsed UV-light has a potential for inactivation of milk pathogens.