Fermented sour soup is a unique traditional sour food of the Miao and Dong ethnic groups in China. However, due to the commonly used traditional fermentation, the unstable fluctuation of microbial community often leads to some unpleasant inherent off-flavours in rice sour soup. In this study, the microbial composition and volatile flavour components of off-flavour and normal rice sour soup were examined by high-throughput sequencing and headspace solid-phase microextraction coupled with gas chromatographymass spectrometry. Then, based on Pearson correlation coefficients, the correlations between fungi and bacteria and those between core microorganisms and key volatile flavour components were investigated. The dominant fungal genera included Candida, Dekkera, Pichia, Rhizopus, and Issatchenkia, whereas the dominant bacterial genera included Lactobacillus, Pectinatus, Bifidobacterium, Pseudomonas, and Acetobacter. Compared to normal rice sour soup, off-flavoured rice sour soup contained the significantly increased relative abundance of Candida and the significantly decreased relative abundance of Dkkera. In addition, 80 volatile flavour compounds detected in off-flavoured rice sour soup, mainly including esters, acids, and alcohols, were different from those detected in normal rice sour soup. Especially, some substances were only detected in off-flavoured rice sour soup, such as 1-hexanol (68–207 μg/kg), butyric acid (20–92 μg/kg) and heptanoic acid (12–45 μg/kg), which might be the main source of off-flavours. These results provide new ideas and strategies of removing or abating odour from fermented rice sour soup.