The recent discovery of nitrous oxide (N2O)-reducing bacteria suggests a potential biological sink for the potent greenhouse gas N2O. For an application toward N2O mitigation, characterization of more isolates will be required. Here, we describe the successful enrichment and isolation of high-affinity N2O-reducing bacteria using a N2O-fed reactor (N2OFR). Two N2OFRs, where N2O was continuously and directly supplied as the sole electron acceptor to a biofilm grown on a gas-permeable membrane, were operated with acetate or a mixture of peptone-based organic substrates as an electron donor. In parallel, a NO3 - -fed reactor (NO3FR), filled with a nonwoven sheet substratum, was operated using the same inoculum. We hypothesized that supplying N2O vs NO3 - would enhance the dominance of distinct N2O-reducing bacteria. Clade II type nosZ bacteria became rapidly enriched over clade I type nosZ bacteria in the N2OFRs, whereas the opposite held in the NO3FR. High-throughput sequencing of 16S rRNA gene amplicons revealed the dominance of Rhodocyclaceae in the N2OFRs. Strains of the Azospira and Dechloromonas genera, canonical denitrifiers harboring clade II type nosZ, were isolated with high frequency from the N2OFRs (132 out of 152 isolates). The isolates from the N2OFR demonstrated higher N2O uptake rates (Vmax: 4.23 × 10-3-1.80 × 10-2 pmol/h/cell) and lower N2O half-saturation coefficients (Km,N2O: 1.55-2.10 μM) than a clade I type nosZ isolate from the NO3FR. Furthermore, the clade II type nosZ isolates had higher specific growth rates on N2O than nitrite as an electron acceptor. Hence, continuously and exclusively supplying N2O in an N2OFR allows the enrichment and isolation of high-affinity N2O-reducing strains, which may be used as N2O sinks in bioaugmentation efforts.