Quorum sensing inhibitors (QSI) may provide new research direction for the defense of bacterial diseases and biofilm related marine biofouling. QSI screening and development from natural source have received great interest in recent years. In this study, 17 marine bacterial strains, which were found exhibiting anti-quorum sensing activity in previous study, were secondary screened using pigment inhibition assay. One active bacterium 108 was identified as Staphylococcus saprophyticus based on the 16S rDNA sequence analysis and morphological observation. The culture condition of bacterium 108 was optimized. The results showed that moderate pH (6.0-7.0) and temperature (25°C) favored the bacterial growth and bioactive metabolites production. Yeast extracts content was the most importantmediumcomponent affecting the bacterial growth and bioactive metabolites production. By culturing under the optimized condition, the anti-quorum sensing activity of S. saprophyticus 108 and the yield of crude extracts was increased about 50% and 1.5 times, respectively, compared to the bacterial cultures before optimization. The active metabolites in S. saprophyticus 108 cultures were further extracted, isolated and identified. Cyclo (Pro-Leu), was identified from the active subfraction of the bacterium by GC-MS analysis. It was found for first time that Cyclo (Pro-Leu) exhibitedmoderate anti-quorumsensing activity.