AbstractThis study investigated the interactions of four bacteria strains isolated from Yamahai‐shubo, the source of yeast used to produce a Japanese traditional rice wine, Yamahai‐shikomi sake. The bacterial strains were nitrate‐reducing Pseudomonas sp. 61‐02, Leuconostoc mesenteroides LM‐1, Lactiplantibacillus plantarum LP‐2, and Latilactobacillus sakei LS‐4. We examined fermentation factors for Yamahai‐shubo and Yamahai‐shikomi sake samples to compare the suitability of their bacterial combination (16 variations). As a result of principal component analysis, we found that two major groups were formed; one containing strain LP‐2 and the other containing strain LS‐4, and that strains LP‐2 and LS‐4 were important in the Yamahai‐shikomi sake in the presence of strains 61‐02 and LM‐1. Then, we investigated the effects of strains LP‐2 and LS‐4 on the concentration of organic acids (pyruvic acid, citric acid, succinic acid, malic acid, and lactic acid) in Yamahai‐shikomi sake. Only in lactic acid, a tendency to decrease with a smaller proportion of LS‐4 strains in Yamahai‐shubo was observed. Subsequently, their effect on the concentration of diacetyl, crucial for aroma, was investigated between the LP‐2 and LS‐4 strains. The sample prepared in the absence of strain LS‐4 exhibited the lowest concentration of diacetyl. This result was supported by the statistical analysis for the sensory scores performed for aroma of each Yamahai‐shikomi sake sample. In conclusion, strain LP‐2 plays a more significant role in improving Yamahai‐shikomi sake quality with strains LM‐1 and 61‐02 rather than strain LS‐4 in Yamahai‐shubo preparation and Yamahai‐shikomi sake brewing.