A metagenomic analyses of meju revealed filamentous fungal strains from traditional meju. To confirm their characteristics, total of 30 species filamentous fungi in 7 genera, including Aspergilli, Penicilli, Cladosporia, and Mucor, were isolated and identified, and determinated the production of mycotoxins. The results of metagenome analysis showed that the distribution and abundance of fungi were very diverse according to the difference in production regions and fermentation conditions of meju. The activity of peptidase, amylase, and lipase was investigated in nonaflatoxigenic Aspergillus flavus/oryzae among strains isolated from meju, and this activity was compared with commercially available yellow-koji strains for meju production. Nonaflatoxigenic A. oryzae strain AO3222 showed more suitable enzyme activity for fermentation of meju than conventional yellow-koji. Also, result of whole genome analysis, it was found that all yellow-koji strains were clustered in very similar close relationships, thus they had almost identical genomic characteristics. However, strain AO3222 was clustered into A. oryzae group different from yellow-koji. To analyze the possibility of using the AO3222 strain as a new koji for the production of soybean meju, the AO3222 strain was inoculated and fermented simultaneously with strains such as A. flavus, Mucor racemosus, and Rhizopus arrhizus. Through this, the ability to reduce the production of aflatoxin in AO3222 strains was analyzed, and the distribution pattern of each strain inoculated in meju was analyzed using the metagenomic method.