Reactivity-controlled compression ignition (RCCI) combustion burns fuels of varying reaction rates to enhance combustion stratification. Biogas-diesel RCCI combustion requires energy assessment to improve power and reduce emissions. This study investigates the effects of different carbon dioxide (CO2) contents (25, 35, and 45) and port mixing distances (0, 28.75, 57.5, 86.25, and 115 mm) experimentally, at 6.5 bar IMEP and 1600 rpm in a premixed and port injection at the valve. An exergy analysis facilitates understanding high-CO2 biogas stratification and the cause of emissions trade-off experimentally, unavailable in the literature. Based on energy analysis, a 35 CO2 content demonstrates moderate combustion work and enhanced output power, while 45 CO2 shows lower heat loss across the mixing distances. Injecting biogas at the valve increases combustion work by 9.11 � 27.33 and reduces power output by 0.33 � 4.90 due to increased exhaust loss. Based on exergy analysis, evenly distributed in-cylinder temperatures and sufficient fuel stratification were observed for 35 CO2 and 50 distance. The 35 CO2 increases the internal energy potential recovery by 24.26 over mixing spaces because of reduced destruction. Injection at the valve causes stratified CO2 layers at the bowl centerline and diffused temperature at the cylinder wall, simultaneously decreasing CO2, carbon monoxide, unburned hydrocarbon, nitrogen oxides, and particulate matter emissions by 4.46, 2.04, 3.05, 44.67, and 9.13, respectively. Therefore, increased compression work allows more heat transfer through the exhaust, while heat outflow through the cylinder wall reduces combustion work. Furthermore, the reduced emissions due to increased CO2 content at higher mixing distances compromised the output power of the biogas-diesel RCCI engine. © 2021 Elsevier Ltd