In this study, 424 actinomycete strains isolated from soil and litter samples on Cat Ba island (Haiphong, Vietnam) were subjected to the screening for the inhibitory activities against microorganisms, including bacteria (Micrococcus luteus, and Escherichia coli) and eukaria (Candida albicans and Fusarium oxysporium). Through two screening steps, 17 strains were selected for their high inhibitory activity against one or more target microorganisms. Crude extracts in ethyl acetate from culturing media of the selected strains were analyzed via thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC), in which chloramphenicol, kitasamycin, erythromycin and raw extract of anthracycline were used as standards. The obtained results showed that antibiotic substances produced by the selected strains could not be put in any group of the analyzed standards, except the strain A396 which appeared to produce a chloramphenicol-like antibiotic. Taxonomical studies based on the morphology and 16S rDNA sequencing indicated that the collection of actinomycetes isolated from Catba island contained mainly Streptomyces species (about 70 percent) and the group of rare actinomycetes (non-Streptomyces) which made of 30 percent of the collection was dominated by Micromonospora, Nonomureae and Nocardia genera. Of the 17 selected strains with highest antimicrobial activity, ten strains were affiliated to the genus Streptomyces (as based on the morphology) and seven strains belonged to the genus Nonomuraea (as based on 16S rDNA sequence analyses). The strains selected in this study could serve as sources for discovering new antibiotic substances in Vietnam.