AbstractThis study investigated the interactions of four bacteria strains isolated fromYamahai‐shubo, the source of yeast used to produce a Japanese traditional rice wine,Yamahai‐shikomisake. The bacterial strains were nitrate‐reducingPseudomonassp. 61‐02,Leuconostoc mesenteroidesLM‐1,Lactiplantibacillus plantarumLP‐2, andLatilactobacillus sakeiLS‐4. We examined fermentation factors forYamahai‐shuboandYamahai‐shikomisake 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 theYamahai‐shikomisake 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) inYamahai‐shikomisake. Only in lactic acid, a tendency to decrease with a smaller proportion of LS‐4 strains inYamahai‐shubowas 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 eachYamahai‐shikomisake sample. In conclusion, strain LP‐2 plays a more significant role in improvingYamahai‐shikomisake quality with strains LM‐1 and 61‐02 rather than strain LS‐4 inYamahai‐shubopreparation andYamahai‐shikomisake brewing.