Background: Antimicrobial substances are mainly produced by bacteria and lower fungi, and have great roles in the treatment of most infectious diseases..Purpose: Production of antistaphylococcal metabolite from Aureobasidium pullulans by development of a cultural medium using response surface methodology. Methods: Production of antistaphylococcal metabolite from Aureobasidium pullulans was optimized in shake flasks using a statistical experimental design approach. Effect of various components in the basal medium, glucose, peptone, KH2PO4 as well as initial pH and temperature were statistically combined using a 2 level, 4 factor experimental design and tested for their influence on maximal antistaphylococcal metabolite production. Results were analyzed using response surface methodology (RSM) software. Results: Optimum production of antistaphylococcal metabolite occurred at glucose 2.0%, peptone 2.5%, KH2PO4 0.15%, pH 4.0 and temperature 30°C. The maximum amount of antistaphylococcal metabolite 900 U/flask from about 0.85 g of dry weight biomass was extracted. Conclusion: The antistaphylococcal activity of Aureobasidium pullulans seemed to be associated with primary metabolite rather than secondary metabolite. However, this conclusion should be taken with caution because both secondary metabolites as well as antibiotics are heterogeneous group and our knowledge regarding the exact definitions and of secondary metabolite / antibiotics are far from the perfection.