Mudstone, a class of sedimentary rocks rich in organic matter, possesses considerable potential for biogas production. Mudstones possess a rich biological origin, which is conducive to refining the mechanisms and enrichment patterns of biogenic gas reservoirs. This has significant theoretical and practical implications for guiding the exploration and development of Quaternary mudstone gas reservoirs. Furthermore, the Qaidam Basin is an excellent place for the geological storage of CO2 due to its rich petroleum reservoir conditions. Experimental research on biogas production under diverse CO2 pressure-mudstone-microorganism-water interactions is conducted to determine the biogas production mechanism of mudstone under different CO2 pressures during sequestration circumstances. According to the results: (1) under supercritical carbon dioxide conditions, there is a slight initial increase in biogas production, followed by a gradual decrease. The periods and peaks of gas production vary among the different reaction groups. As carbon dioxide pressure increases, the gas production cycle lengthens significantly, while the gas yield declines. (2) Siderite and secondary carbonate minerals have increased in the mudstone's mineral fraction both before and after biogas production, while clay mineral groups have decreased. Specifically, there was a notable drop in chlorite and kaolinite. (3) Microorganism species in the system were analyzed, and the results showed that there was a gap in each microorganism's ability to adapt to its surroundings, and the diversity and quantity of bacteria declined with increasing pressure. After carbon dioxide was fluxed, there was a considerable shift in the pattern of biogas generation, which consequently had a major impact on the alterations in mineral fractions.