ABSTRACT Here, a new medium named as intensive soil extract medium (ISEM) based on new soil extract (NSE) using 80% ethanol was used to efficiently isolate previously uncultured bacteria and new taxonomic candidates, which accounted for 49% and 55% of the total isolates examined (n=258), respectively. The new isolates were affiliating with seven phyla such as Proteobacteria, Acidobacteria, Firmicutes, Actinobacteria, Verrucomicrobia, Planctomycetes , and Bacteroidetes . The result of chemical analysis showed that NSE included more diverse components of low-molecular-weight organic substances than two conventional soil extracts using distilled water. Cultivation of previously uncultured bacteria is expected to extend knowledge through the discovery of new phenotypic, physiological and functional properties, and even roles of unknown genes. IMPORTANCE Either metagenomics or single-cell sequencing can detect unknown genes from uncultured microbial strains in environments and may find their significant potential metabolites and roles. However, such gene/genome-based techniques still have a critical problem making impossible for further applications through cultivation. To solve this problem, various approaches for cultivation of uncultured bacteria have been developed, but they still have lack of skill to grow them on solid media for isolation and subculture.