This study evaluated the impacts of a nitrification inhibitor (3,4-dimethylpyrazole phosphate, DMPP) and herbicides (atrazine and glyphosate) on nitrification, gross nitrite, and nitrate (NO2 − -N + NO3 − -N) consumption rate, nitrous oxide (N2O) emission, and abundances of microbial functional genes related to nitrogen (N) cycling in an Australian sugarcane soil. The experiment included four treatments: blank control (CK), DMPP application (NI), atrazine application (ATR), and glyphosate application (GLY). All treatments received (NH4)2SO4 at 50 mg N kg−1 dry soil and KNO3 at 50 mg N kg−1 dry soil and were incubated initially at 55% of water holding capacity (WHC) for 7 days and subsequently at 75% WHC for another 7 days (K15NO3 with 5 atom% 15N added at the beginning of each stage). Compared with the CK treatment, DMPP application significantly decreased N2O emissions throughout the incubation, while atrazine or glyphosate application significantly inhibited N2O emissions only during the 4–7-day period. DMPP application also decreased ammoniumoxidizing bacteria (AOB) amoA gene abundances, gross NO2 − -N + NO3 − -N consumption rates at 55 and 75% WHC, and nirS and nirK gene abundances of denitrifiers at 75% WHC. The atrazine and glyphosate applications decreased the gross nitrification and NO2 − -N + NO3 − -N consumption rates, abundances of both ammonium-oxidizing archaea (AOA) and AOB amoA genes at 55 and 75% WHC, and abundances of functional genes related to different reactions of the denitrification during the incubation. These results suggested that DMPP, atrazine, and glyphosate could decrease soil gross nitrification and denitrification rates perhaps by inhibiting microbial functional gene abundances and that application of DMPP could effectively reduce N2O emissions in the sugarcane cropping soil.