The goal of this study was to elucidate the mechanisms of nitrous oxide (NO) production from a bioreactor for partial nitrification (PN). Ammonia-oxidizing bacteria (AOB) enriched from a sequencing batch reactor (SBR) were subjected to NO production pathway tests. The NO pathway test was initiated by supplying an inorganic medium to ensure an initial NH-N concentration of 160 mg-N/L, followed by NO (20 mg-N/L) and dual NHOH (each 17 mg-N/L) spikings to quantify isotopologs of gaseous NO (NO, NO, and NO). NO production was boosted by NHOH spiking, causing exponential increases in mRNA transcription levels of AOB functional genes encoding hydroxylamine oxidoreductase (haoA), nitrite reductase (nirK), and nitric oxide reductase (norB) genes. Predominant production of NO among NO isotopologs (46% of total produced NO) indicated that coupling of NHOH with NO produced NO via N-nitrosation hybrid reaction as a predominant pathway. Abiotic hybrid NO production was also observed in the absence of the AOB-enriched biomass, indicating multiple pathways for NO production in a PN bioreactor. The additional NO pathway test, where NH was spiked into 400 mg-N/L of NO concentration, confirmed that the hybrid NO production was a dominant pathway, accounting for approximately 51% of the total NO production.