Organic compounds and mineral nitrogen (N) usually increase nitrous oxide (N2O) emissions. Vinasse, a by‐product of bio‐ethanol production that is rich in carbon, nitrogen, and potassium, is recycled in sugarcane fields as a bio‐fertilizer. Vinasse can contribute significantly to N2O emissions when applied with N in sugarcane plantations, a common practice. However, the biological processes involved in N2O emissions under this management practice are unknown. This study investigated the roles of nitrification and denitrification in N2O emissions from straw‐covered soils amended with different vinasses (CV: concentrated and V: nonconcentrated) before or at the same time as mineral fertilizers at different time points of the sugarcane cycle in two seasons. N2O emissions were evaluated for 90 days, the period that occurs most of the N2O emission from fertilizers; the microbial genes encoding enzymes involved in N2O production (archaeal and bacterial amoA, fungal and bacterial nirK, and bacterial nirS and nosZ), total bacteria, and total fungi were quantified by real‐time PCR. The application of CV and V in conjunction with mineral N resulted in higher N2O emissions than the application of N fertilizer alone. The strategy of vinasse application 30 days before mineral N reduced N2O emissions by 65% for CV, but not for V. Independent of rainy or dry season, the microbial processes were nitrification by ammonia‐oxidizing bacteria (AOB) and archaea and denitrification by bacteria and fungi. The contributions of each process differed and depended on soil moisture, soil pH, and N sources. We concluded that amoA‐AOB was the most important gene related to N2O emissions, which indicates that nitrification by AOB is the main microbial‐driven process linked to N2O emissions in tropical soil. Interestingly, fungal nirK was also significantly correlated with N2O emissions, suggesting that denitrification by fungi contributes to N2O emission in soils receiving straw and vinasse application.