The reaction kinetics of lithotrophic ammonia-oxidizing bacteria (AOB) are strongly dependent on dissolved oxygen (DO) as their metabolism is an aerobic process. In this study, we estimate the kinetic parameters, including the oxygen affinity constant (Km[O2]) and the maximum oxygen consumption rate (Vmax[O2]), of different AOB species, by fitting the data to the Michaelis-Menten equation using nonlinear regression analysis. An example for three different species of Nitrosomonas bacteria (N. europaea, N. eutropha, and N. mobilis) in monoculture is given, finding a Km[O2] of 0.25±0.05mg l-1, 0.47±0.09mg l-1, and 0.28±0.08mg l-1, and a Vmax[O2] of 0.07±0.04 pg h-1cell-1, 0.25±0.06 pg h-1cell-1, and 0.02±0.001 pg h-1cell-1 for N. europaea, N. eutropha, and N. mobilis, respectively. This study shows that of the analyzed AOB, N. europaea has the highest affinity towards oxygen and N. eutropha the lowest affinity towards oxygen, indicating that the former can convert ammonia even under low DO conditions. These results improve the understanding of the ecophysiology of AOB in the environment. The accuracy of mathematically modelled ammonia oxidation can be improved, allowing the implementation of better management practices to restore the nitrogen cycle in natural and engineered water systems.