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Terrestrial actinobacteria have served as a primary source of bioactive compounds; however, a rapid decrease in the discovery of new compounds strongly necessitates new investigational approaches. One approach is the screening of actinobacteria from marine habitats, especially the members of the genusStreptomyces. Presence of this genus in a marine sponge,Haliclonasp., was investigated using culture-dependent and -independent techniques. 16S rRNA gene clone library analysis showed the presence of diverseStreptomycesin the sponge sample. In addition to the dominant genusStreptomyces, members of six different genera were isolated using four different media. Five phylogenetically new strains, each representing a novel species in the genusStreptomyceswere also isolated. Polyphasic study suggesting the classification of two of these strains as novel species is presented. Searching the strains for the production of novel compounds and the presence of biosynthetic genes for secondary metabolites revealed seven novel compounds and biosynthetic genes with unique sequences. In these compounds, JBIR-43 exhibited cytotoxic activity against cancer cell lines. JBIR-34 and -35 were particularly interesting because of their unique chemical skeleton. To our knowledge, this is the first comprehensive study detailing the isolation of actinobacteria from a marine sponge and novel secondary metabolites from these strains.