This study has been carried out to optimize an antimicrobial activity of flavonoid extract from pomelo peels against Staphyloccus aureus (S. aureus). A comparative analysis of total phenolic content (TPC), total flavonoid content (TFC) and antioxidant activity were done on two parts of peel which are albedo (inner peel) and flavedo (outer peel). Based on the result obtained, flavedo showed higher TPC, TFC and antioxidant activity (304.20 mg /g, 74.30 mg /g and 46.86 % respectively) when compared to albedo (150.98 mg /g, 52.97 mg /g and 24.70 % respectively). The effects of different extract concentration and pH on inhibition zone of S. aureus were optimized using Research Surface Methodology (RSM). The optimal condition of parameters was obtained based on the maximum zone of S. aureus inhibition at extract concentration of 200 mg/mL and pH of 4.8. The antimicrobial film has been developed by using optimal conditions by incorporating the flavonoid extract into chitosan polymer.