Anthropogenic disturbances to nitrogen (N) cycling, particularly through agricultural N use, have intensified nitrous oxide (N2O) emissions. Developing wheat lines with biological nitrification inhibition (BNI) is a promising strategy to reduce such emissions. However, the effectiveness of BNI depends not only on plant characteristics, but also on how they interact with environmental factors such as soil pH and elevated atmospheric CO2 (eCO2). This study evaluated the response of a BNI-capable wheat line (Triticum aestivum cv. ROELFS) to eCO2 under contrasting soil pH during the 30 days following ammonium fertilization. Two wheat lines (ROELFS-Control and ROELFS-BNI) were grown under ambient (400 ppm) and elevated (800 ppm) CO2 in acidic (pH 5.3) and alkaline (pH 8.8) soils. N2O emissions, nitrifying and denitrifying microbial communities, and soil chemical properties were monitored to assess plant-soil-microbe interactions. ROELFS-BNI consistently reduced N2O emissions under all conditions by reducing archaeal (Nitrososphaeraceae) and bacterial nitrifiers (Nitrosomonadaceae, Nitrospiraceae) without major shifts in overall microbial composition, indicating high specificity of BNI exudates. Elevated CO2 effects on N2O emissions were pH-dependent. In acidic soil, eCO2 increased emissions in ROELFS-Control but not in ROELFS-BNI, likely due to the enrichment of complete denitrifiers (e.g. Rhodanobacteraceae). However, in alkaline soil, eCO2 reduced N2O emissions in both Control and BNI lines, especially the latter, which was associated with a higher abundance of N2O-reducing denitrifiers (e.g. Burkholderiaceae). This study highlights the potential of ROELFS-BNI wheat as a sustainable practice to mitigate N pollution adaptable to diverse soils and predicted CO2 atmospheric conditions.