Actinomycetesare an important source of microorganisms used in medicine. Tens of thousands of biologically active substances of microbial origin have been found, about 45% of which are produced by actinomycetes. The marine environment has high salinity, high pressure, low temperature, low nutritive content and other characteristics, so that the population characteristics and metabolic pathways of marine actinomycetes are different from those of terrestrial actinomycetes, and can be a source of novel species and metabolites. In total, 165 antimicrobial isolates were screened from13 marine sediment samples collected in the seas around Dalian using nine different media. The results showed that improved humic acid vitamin agar medium (HV-2) and glycerol arginine agar medium (M2) were the best types of medium for isolating marine actinomycetes, followed by improved LSE-SE-2 agar medium (LSE-SE-2) and oat agar medium (OM). Using agar block methods, the antagonistic activity of the 165 strains against three pathogens (Fusariumoxysporium f sp. cucumarinum, Staphylococcus aureus and Escherichia coli) was analyzed in vitro. Among the 165 isolates, 95 strains showed antagonistic activity against these three test pathogens. Of the 165 isolates, 85 (51.5%) strains showed activity against S.aureus. The diameter of the inhibition zone of four of the 85 strains was more than 30 mm; 27 strains (16.4% of the total isolates)showed activity against E. coli, and the diameter of the inhibition zone of strains YH6 and YH21 against E. coli was more than 27mm. Six strains (M95, M108, HV14, HL9, HE16,HE66), 3.6% of the total isolates, showed activity against F. oxysporumf sp. cucumarinum. The results indicated that marine actinomycetes often show antagonistic activity against bacteria, especially Gram-positive bacteria.Meanwhile, 16S rDNA sequences of interesting strains with either special phenotypes or strong antimicrobial activity were analyzed.Phylogenetic analysis using 16S rDNA sequencing revealed that marine Streptomyces showed abundant species diversity and broad activity. Apart from the hygroscopicus group,95 marine Streptomyces showed antagonistic activity covering all groups of Streptomyces. The 95 marine Streptomyces belong to different groups such as the roseosporusgroup, golden group, albosporus group, cinereus group, yellow group, blue group,lavendulae group, black group, griseorubroviolaceus group, griseofuscus group and green group. According to the SciFinder substances library, the biological activity of recorded species from the NCBI library, which had sequences approximately homologous to the 95 marine Streptomyces isolates, was analyzed. The 95 marine Streptomyces showed bioactivity, but 23 strains among the recorded species failed to exhibit activity against the pathogens. Meanwhile, 13 isolates from the 95 marine Streptomyces exhibited bioactivity, whereas the 13 corresponding recorded species failed to exhibit activity. This suggested that different habitats give the same species different activities. The same species isolated from marine sediments and soil showed different activities. Streptomyces species from marine sediments often showed antagonistic activity against Gram-positive bacteria, and had low antagonistic activity against fungi and Gram-negative bacteria.Through the analysis of the antagonistic activity of marine Streptomyces, the results will help researchers to improve our understanding of microbial diversity,and the isolates obtained will also help us to search for metabolites with novel activity.