A taxonomic study on 77 actinomycete isolates from 16 peat swamp forest soils samples collected in Rayong Province was carried out. The actinomycete isolates were divided in 9 genera including Streptomyces (35 isolates), Micromonospora (3 isolates), Microbispora (9 isolates), Microtetraspora (6 isolates), Actinomadura (2 isolates), Nocardia (4 isolates), Actinoallomurus (1 isolate), Dactylosporangium (3 isolates) and unknown species (13 isolates) based on their morphology. On the basis of 16S rRNA gene sequence analyses for the representative isolates and the polyphasic approach, the novel species, isolate RY35-23 and RY35-68 were closely related to the type strains with 98.94 and 97.5 % 16S rRNA gene sequence similarity. The name Dactylosporangium sucinum and Actinomadura rayongensis were proposed for them, respectively. In the screening of antimicrobial activities of the isolated actinomycetes 41 isolates in genera Streptomyces (25 isolates), Micromonospora (1 isolate), Microtetraspora (1 isolate), Microbispora (1 isolate), Dactylosporangium (2 isolates), Actinoallomurus (1 isolate) and unknown species (5 isolates) showed antimicrobial activities against tested microorganisms. Chromatographic and liquid chromatography/mass spectrometry revealed that isolate RCU-197 produced actinomycin D as the active compound against Gram-positive bacteria. Based on 16S rRNA gene sequence analyses, isolate RCU-197 showed the characteristics of the novel actinomycete species that it is required for further study.