The complex structural and functional responses of agricultural soil microbial communities to the addition of carbonaceous compounds such as biochar remain poorly understood. This severely limits the predictive ability for both the potential enhancement of soil fertility and greenhouse gas mitigation. In this study, we utilized shotgun metagenomics in order to decipher changes in the microbial community in soil microcosms after 14 days of incubation at 23°C, which contained soils from biochar-amended and control plots cultivated with Napier grass. Our analyses revealed that biochar-amended soil microbiomes exhibited significant shifts in both community composition and predicted metabolism. Key metabolic pathways related to carbon turnover, such as the utilization of plant-derived carbohydrates as well as denitrification, were enriched under biochar amendment. These community shifts were in part associated with increased soil carbon, such as labile and aromatic carbon compounds, which was likely stimulated by the increased available nutrients associated with biochar amendment. These findings indicate that the soil microbiome response to the combination of biochar addition and to incubation conditions confers enhanced nutrient cycling and a small decrease in CO2 emissions and potentially mitigates nitrous oxide emissions.IMPORTANCE The incorporation of biochar into soil is a promising management strategy for sustainable agriculture owing to its potential to sequester carbon and improve soil fertility. Expanding the addition of biochar to large-scale agriculture hinges on its lasting beneficial effects on the microbial community. However, there exists a significant knowledge gap regarding the specific role that biochar plays in altering the key biological soil processes that influence plant growth and carbon storage in soil. Previous studies that examined the soil microbiome under biochar amendment principally characterized only how the composition alters in response to biochar amendment. In the present study, we shed light on the functional alterations of the microbial community response 2 years after biochar amendment. Our results show that biochar increased the abundance of genes involved in denitrification and carbon turnover and that biochar-amended soil microcosms had a reduction in cumulative CO2 production.