Biochar can affect CO2 emission and C sequestration from soils, but little is known about the effects of its re-application and interaction with easily accessible substrates. Since most agricultural soils are continuously reworked, understanding the mechanics of CO2 evolution as affected by soil amendments and their combinations may have important lessons for the global effort to combat climate change. In this study carried out in a controlled environment, we tested the short-term effects of biochar, and its re-application at different rates, on the production of CO2 emission and C accumulation in samples of arable soils. We used a loamy Haplic Luvisol as the substrate and added varying amounts of biochar and sources of easily accessible N and C. We observed CO2 evolution for 20 days at optimal temperature and moisture conditions. We found that in the control treatment with no biochar, the total potential respiration after the addition of (NH4)2SO4 (N) and glucose (G) was increased compared to basal respiration. The addition of biochar reduced CO2 emission in the control, N- and G-stimulated treatments by 12–22, 13‒24 and 2‒21%, respectively. Conversely, the application of biochar increased CO2 emission in the combined NG treatment. Application of biochar at a higher rate, as well as its re-application, increased soil organic carbon content and reduced emission of CO2 into the atmosphere.