Total of 178 Streptomyces isolates were obtained from soil samples collected in Wiangsa District, Nan province, Thailand. Initial grouping based on morphological, physiological and biochemical characteristics resulted in overlapping groups and distinctive groupings could not be obtained. Therefore, grouping based on analysis of DNA fingerprinting of restriction fragment length polymorphism (RFLP) of 16S-lTS digested with restriction enzyme Haelll was performed. Isolated strains were classified into 11 groups with similar DNA fingerprint patterns. In addition, RFLP DNA fingerprints using BstUl digestion gave more diversed patterns and 39 subgroups were obtained. One representative strain for each of the 39 groups were further analyzed for 16S rDNA sequences, and phylogenetic tree was constructed. Fourteen clusters were obtained from phylogenetic tree. By comparison of the grouping results from the 16S-ITS RFLP with 16S rDNA sequences, the 16S-ITS RFLP fingerprinting provided a higher resolution than 16S rDNA sequencing-based analysis. These results indicated that 16S-ITS RFLP fingerprinting technique was effective in studying classification and characterization the level of species in Streptomyces. Moreover, screening for Streptomyces spp. Capable of producting antimicrobial compounds was performed. Fiveteen isolates were found to have antifungal activity while 10 isolates produced antibacterial compounds. In conclusion, grouping and characterization of Streptomyces spp. Isolated from soil samples collected from just one district of Thailand were highly diversed and could be used for biotechnological exploitation of these bacteria.