Traditional grain vinegar is fermented using multiple acetic acid bacteria (AAB) at various temperatures. A single AAB showed high acid–producing ability at 30 °C with a 5% alcohol concentration and an initial pH adjusted to 4.0. Multiple AAB were similar to single AAB, but the optimal initial pH was 3.0. Acid production ability according to the type of AAB was higher in multiple AAB than that for single AAB. That is, using multiple AAB helped to increase the titratable acidity of traditional grain vinegar. In addition, increasing the titratable acidity and content of volatile flavor compounds was advantageous when two, rather than four, AAB types were mixed and used. Titratable acidity was high at medium temperatures (30 °C), but volatile flavor compounds increased at low temperatures (20 °C) under multiple AAB. A 16S rDNA–based microbiome taxonomic profiling analysis identified differences in beta diversity due to multiple AAB and fermentation temperatures. In particular, beta diversity analysis revealed a specific pattern when a mixture of Acetobacter ascedens GV–8 and Acetobacter pasteurianus GV–22 were fermented at low temperature (20 °C). Therefore, we propose the application of multiple AAB with acidic and flavor–producing properties in traditional grain vinegar.