Abstract: To search for natural and efficient anti-fouling active substances， 4 sponge-associated microorganisms， which were preliminarily found with strong activities of restraining diatom attachment， were selected to verify and compare their activities. The most active bacterial strain of UST050418-715 was selected based on the activities against the settlement of 3 tested diatoms， including Nitzschia closterium， Amphora coffeaeformis， and Nitzschia frustulum. And the strain UST050418-715 was identified as Bacillus pumilus based on the 16S rDNA sequence analysis， cell morphology and colony characteristics. To optimize the cultivation conditions of strain UST050418-715， orthogonal tests were carried out with 4 factors （yeast extract concentration， peptone concentration， NaCl concentration and pH） and 3 levels. The 4 factors all affected the anti-settlement activities of fermentation products， and interferential efficiencies of which are yeast concentration > peptone concentration > NaCl concentration > pH.Combined the results of orthogonal tests with crude exact yields and growth of the strain， the optimum culture conditions of UST050418-715 were concluded as marine 2216E medium containing 7.5 g/dm³ peptone， 3 g/dm³ yeast extract， 10.45 g/dm³ NaCl， and pH 9.5.