Streptomyces anulatus NEAE-94, an antagonistic actinomycete newly isolated from Egyptian soil, exhibited a broad antimicrobial spectrum against several microorganisms including multidrug-resistant Staphylococcus aureus, E. coli, Bacillus subtilis, Pseudomonas aeruginosa and Candida albicans. Fifteen factors (starch, KNO,, K2HPO4, yeast extract, NaCl, MgSO4, CaCO3, FeSO4, pH, temperature, agitation speed, medium volume, inoculum size, inoculum age and fermentation time) were examined for their significances on production of antimicrobial metabolites using Plackett-Burman design. Among the variables screened, agitation speed, inoculum size and inoculum age had significant effects on antimicrobial activities production. These factors were further optimized using Box Behnken statical design.The optimal conditions achieved were high level of agitation speed (250 rpm min 1), middle level of inoculum size (4%) and low level of inoculum age. The Antagonistic activity produced from the optimized culture conditions against multidrug-resistant Staphylococcus aureus, showed about two fold increase than that obtained from the un-optimized medium. As a result, a medium of the following formula was predicted to be the optimum for producing an extracellular antimicrobial metabolites in the culture filtrate of Streptomyces anulatus NEAE-94 L-1: Starch 20 g, KNO3, 2 g, K2HPO, 0.5 g,NaCl 0.1 g, MgSO4.7H20 0.1 g, CaCO3 3 g, FeSO4.7H20 0.01 g, pH 6.5, temperature 25°C, agitation speed 250 rpm mm1, medium volume 75 mL, inoculum size 4%,v/v, inoculum age 60 h and fermentation period 5 days.