The use of a metal mesh device (MMD) as a precision bacterial separation filter is described. The MMD uses a structure in which identically shaped pores are arranged in a thin metal membrane. Four types of MMD with different pore sizes were used to fractionate bacteria in two types of soil. Through metagenomic analysis, the distribution of bacteria in the soil samples and in each MMD fraction was examined. In addition, eight types of previously described organic compound-degrading bacteria were used to evaluate the method, and changes in their composition following MMD fractionation were investigated. It was found that MMD fractions were enriched for all eight bacteria when compared with the initial sample. These results suggest that bacterial fractionation using MMD can enrich bacteria occurring at low frequencies in environmental samples.