Oleuropein and its hydrolysis products are olive phenolic compounds that have antimicrobial effects on a variety of pathogens, with the potential to be utilized in food and pharmaceutical products. While the existing research is mainly focused on individual genes or enzymes that are regulated by oleuropein for antimicrobial activities, little work has been done to integrate intracellular genes, enzymes and metabolic reactions for a systematic investigation of antimicrobial mechanism of oleuropein. In this study, the first genome-scale modeling method was developed to predict the system-level changes of intracellular metabolism triggered by oleuropein in Staphylococcus aureus, a common food-borne pathogen. To simulate the antimicrobial effect, an existing S. aureus genome-scale metabolic model was extended by adding the missing nitric oxide reactions, and exchange rates of potassium, phosphate and glutamate were adjusted in the model as suggested by previous research to mimic the stress imposed by oleuropein on S. aureus. The developed modeling approach was able to match S. aureus growth rates with experimental data for five oleuropein concentrations. The reactions with large flux change were identified and the enzymes of fifteen of these reactions were validated by existing research for their important roles in oleuropein metabolism. When compared with experimental data, the up/down gene regulations of 80% of these enzymes were correctly predicted by our modeling approach. This study indicates that the genome-scale modeling approach provides a promising avenue for revealing the intracellular metabolism of oleuropein antimicrobial properties.