Recent studies on anaerobic digestion (AD) off-gas valorization have explored ways to enhance biological methane production by enriching the digestate with carbon dioxide (CO2). This approach aims to increase AD profitability by biological conversion of CO2 to methane (CH4). However, existing research lacks consensus on the effectiveness of CO2 injection, with the underlying biological mechanisms remaining poorly understood. This study investigates the impact of CO2 injection on AD in a pilot-scale setup using standard on-farm feedstocks. Two pilot-scale reactors were operated in parallel: one serving as a control, and the other featuring CO2 injection through a bubble column with digestate recirculation. Results showed no demonstrable improvement in biomethane yields from CO2 enrichment. In fact, a high-rate CO2 injection had a detrimental effect on methane production.