N2O emissions resulting from the application of nitrogen (N) fertilizers and vinasse represent the main sources of greenhouse gases (GHG) emissions in sugar-energy sector. Conversely, the application of biochar in soils has been worldwide recognized as an strategy to mitigate N2O emissions, although little is known about their effects on soils under energy cane production. The study aimed to evaluate the effects of biochar addition as a strategy to mitigate soil N2O emissions in soil under energy cane cultivation, as well as to quantify the abundance of N2O-producing and N2O-reducing microbial guilds. A greenhouse experiment was conducted in a completely randomized design, with five treatments and four replications. The treatments were: i) no N fertilization (control); ii) N fertilization; iii) N fertilization plus vinasse; iv) N fertilization plus v) N fertilization plus vinasse plus biochar. All treatments (except control) were balanced to receive the same amount of nutrients. Biochar was added at a rate of 5 g kg � 1 of soil. Soil N2O emissions were quantified by static chambers for 78 days, and soil sampling were performed to determine chemical and microbiological attributes, including functional genes of the nitrogen cycle (AOA, AOB, nirK, nirS, nosZI and nosZII) by real-time PCR. Results indicated that vinasse addition increased N2O emissions. Conversely, the application of biochar reduced N2O emissions associated with the application of N fertilizer (56 %) and N fertilizer + vinasse (41 %). The high N2O emissions observed in vinasse treatment were directly correlated with nitrifier microorganisms (AOB and AOA), indicating that nitrification should be the main pathway of N2O emissions in this treatment. The production of energy cane biomass was similar between N fertilizer treatments. High N2O emission intensities (mg N2O g biomass -1) were obtained in treatments with vinasse application. This study concluded that biochar is an effficient strategy to mitigate N2O emissions, providing the first insights into how biochar affects the microbial community associated with N2O emissions from soil under energy cane cultivation.