The increasing atmospheric CO2 concentration is a global concern that affects the plant-bacteria-soil system. Previous studies have investigated plant growth and bacteria activity under CO2 enrichment. However, the effects of coupled elevated CO2 and biochar amendment on the interactions of soil and medicinal plants are not well understood. This study aims to investigate the medicinal plant-soil hydraulic interactions and rhizosphere bacteria communities under coupled CO2 enrichment and biochar conditions. Two levels of CO2 concentration (400, 1000 ppm) and two biochar dosages (3%, 5% by mass) were considered. Pseudostellaria heterophylla was used as the tested medicinal plant. During plant growth, coupled CO2 enrichment and biochar at 3% and 5% dosage increased the volumetric water content at a matric suction of 33 kPa by 97% and 82% respectively, which indicates enhanced water retention. The transpiration rate of P. heterophylla was slightly reduced by 11–30% with an increase in biochar dosage due to higher total suction, while it was significantly reduced by up to 57% due to CO2 enrichment. In the rhizosphere of P. heterophylla, elevated CO2 (1000 ppm) coupled with 3% biochar dramatically increase the relative abundance of Thaumarchaeota, which played an important role in C and N cycles. Moreover, coupled CO2 enrichment and biochar addition resulted in the highest bacterial richness, while 3% biochar at ambient CO2 induced the highest bacterial diversity. This study provides a basis for understanding the medicinal plant-bacteria-soil system under CO2 enrichment and biochar conditions.