Abstract: The newly isolated actinomycetes strain namely M19 was isolated from marine sediment and exhibited antibacterial activity against multidrug-resistant Staphylococcus epidermidis. It was identified on the basis of morphological, cultural, physiological and biochemical properties, together with 16S rRNA sequence as Streptomyces psammoticus strain M19 and sequencing product was deposited in the GenBank database under accession number KT160249. Fifteen factors including starch, KNO3, K2HPO4, yeast extract, NaCl, MgSO4, CaCO3, FeSO4, pH, temperature, agitation speed, medium volume, inoculum size, inoculum age and fermentation time had been examined for their significance on the production of bioactive metabolites using Plackett-Burman design. Out of the fifteen factors; agitation speed, yeast extract, NaCl and KNO3 were selected due to their significant effects on the production of bioactive metabolites and the optimal levels of these variables and the effect of their interactions on bioactive metabolites production were determined by using central composite design. As a result, culture conditions and a medium of the following formula were predicted to be the optimum for production of extracellular bioactive metabolites in the culture filtrate of Streptomyces psammoticus strain M19: Starch 20 g, KNO3 1.5 g, K2HPO4 0.5 g, yeast extract 0.2 g, NaCl 0.5 g, MgSO4 0.1 g, CaCO3 3 g, FeSO4 0.01 g, pH 6.5, temperature 25°C, agitation speed (125 rpm min‾1), medium volume 75 mL, inoculum size 2% (v/v), inoculation age 48 h and fermentation time 5 days. The antagonistic activity produced from the optimized culture conditions against multidrug-resistant Staphylococcus epidermidis, showed about 1.37 fold increase than that obtained from the un-optimized medium.