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Summary: Carbon dioxide (CO2) has the potential to extend the storage time of raw milk, but its industrial application in this regard has not been thoroughly explored. This study aimed to investigate the microflora of CO2‐treated and untreated raw milk from South China using Illumina sequencing. The sequencing results confirmed the presence of Delftia, Pseudomonas and Serratia as the main spoilage species in raw cow milk from South China. However, CO2 treatment was found to selectively inhibit the growth of these spoilage bacteria. Principal coordinate analysis revealed differences in the microbiota structures of samples collected from the Fuyi and Guanyuan sites. Additionally, a total of 119 and 120 bacterial species were identified in the Fuyi and Guanyuan samples, respectively. The spoilage of raw milk from the Fuyi and Guanyuan was delayed by 7 and 19 days, respectively, with the application of CO2 treatment. Conversely, the samples without CO2 treatment were found to be contaminated with Pseudomonas fluorescens and Serratia. Moreover, the CO2‐treated samples from the Fuyi and Guanyuan ranches exhibited a different composition of spoilage bacteria, with a significant increase in the number of lactic acid bacteria (LAB) compared to the control samples. This study suggests that LAB have a remarkable tolerance to CO2, which is consistent with their selection in fruit, vegetable and meat products stored in CO2‐enriched modified atmosphere packaging. The findings of this study provide a foundation for further exploration of the potential application of CO2 treatment in raw milk processing. [ABSTRACT FROM AUTHOR]