This study explored the feasibility of biosurfactant amendment in modifying the interfacial characteristics of carbon dioxide (CO2) with rock minerals under high-pressure conditions for GCS. In particular, while varying the CO2 phase and the rock mineral, we quantitatively examined the production of biosurfactants by Bacillus subtilis and their effects on interfacial tension (IFT) and wettability in CO2-brine-mineral systems. The results demonstrated that surfactin produced by B. subtilis caused the reduction of CO2-brine IFT and modified the wettability of both quartz and calcite minerals to be more CO2-wet. The production yield of surfactin was substantially greater with the calcite mineral than with the quartz mineral. The calcite played the role of a pH buffer, consistently maintaining the brine pH above 6. By contrast, an acidic condition in CO2-brine-quartz systems caused the precipitation of surfactin, and hence surfactin lost its ability as a surface-active agent. Meanwhile, the CO2-driven mineral dissolution and precipitation in CO2-brine-calcite systems under a non-equilibrium system altered the solid substrates, produced surface roughness, and caused contact angle variations. These results provide unique experimental data on biosurfactant-mediated interfacial properties and wettability in GCS-relevant conditions, which support the exploitation of in situ biosurfactant production for biosurfactant-aided CO2 injection.