When investigating the N2O emissions by ammonia oxidizing bacteria, research has mainly focused on identifying and quantifying the biological pathways. This work evaluated previous studies with Nitrosomonas europaea (ATCC 19718) and assessed the role of the abiotic reaction of hydroxylamine with free nitrous acid during transient anoxia. In cultivations when transient anoxia is cyclically imposed, nitrous oxide and hydroxylamine peaked every time upon recovery to aerobic conditions. When using the same culture conditions abiotically (i.e., without biomass, but adding hydroxylamine and nitrite), the volumetric N2O emission rates were very comparable to those from the biological experiments, ranging from 0.04 to 0.08 mg-N/L/h in both abiotic and biotic conditions. These results demonstrate that at the culture conditions tested, abiotically produced N2O is likely the major source of emission. Therefore, for the correct investigation of the biological pathways, abiotic tests must always be performed and hydroxylamine should be added. To our knowledge there is no means to distinguish abiotic from biological N2O production in a biological system, even using N-15 labelling. We suggest that the contribution of abiotic N2O emissions can be minimized by, for example, maintaining lower nitrite concentration and higher pH.