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Objective: The aim of this study was to investigate actinobacterial diversity in Chilean marine sediments., Methods: Actinobacterial diversity in these sediments was investigated by selective isolation method, culture-independent method and phylogenetic analysis based on 16S rRNA gene sequences. Six selective media were used to isolate actinomycetes from sediment samples. The primers for the class Actinobacteria were used for Actinobacterial 16S rRNA gene amplification and then a clone library was constructed for the sediment sample btt. Twenty-two strains with different culture characteristics and 59 clones from sample btt were selected for 16S rRNA gene sequences analysis. To determine requirement for seawater each strain was grown on oatmeal agar prepared with deionized water and with seawater, respectively. Strains were screened for antibiotic activity against bacteria and fungi., Results: In total 328 actinomycetes were obtained. Twenty-two strains which were selected belonged to Streptomyces, Micromonospora, Polymorphospora, Aeromicrobium and Brachybacterium. Fifty-nine clones (40 OTUs) were sequenced, and 60% OTUs belonged to Actinobacteridae, Acidimicrobidae and Rubrobacteridae. The other 40% OTUs, which formed several distinct clades in phylogenetic tree among phylum Actinobacteria may represent new taxonomical groups. 50% of the 47 sea water dependant strains and nineteen strains out of the above 22 strains exhibited antimicrobial activity., Conclusion: There was abundant actinobacterial diversity in the marine sediments of Chile, and the result implied that there were large numbers of unknown actinobacterial groups in the sediments. Actinomycetes from Chilean marine sediments had the potential of producing bioactive secondary metabolites.