Nitrous oxide (N₂O) is a potent greenhouse gas that can be produced by nitrifying and denitrifying bacteria. Yet the effects of N₂O on microbial communities is not well understood. We used batch tests to explore the effects of N₂O on mixed denitrifying communities. Batch tests were carried out with acetate as the electron donor and with the following electron acceptors: nitrate (NO₃⁻), nitrite (NO₂⁻), N₂O, NO₃⁻ + N₂O, and NO₂⁻ + N₂O. Activated sludge from a municipal wastewater treatment plant was used as the inoculum. The bacteria grew readily with N₂O as the sole acceptor. When N₂O was provided along with NO₃⁻ or NO₂⁻, it was used concurrently and resulted in higher growth rates than the same acceptors without added N₂O. The microbial communities resulting from N₂O addition were significantly different at the genus level from those with just NO₃⁻ or NO₂⁻. Tests with N₂O as the sole added acceptor revealed a reduced diversity. Analysis of inferred gene content using PICRUSt2 indicated a greater abundance of genera with a complete denitrification pathway when growing on N₂O or NO₂⁻, relative to all other tests. This suggests that specific N₂O reduction rates are high, and that N₂O alone selects for a low-diversity, fully denitrifying community. When N₂O is present with NO₂⁻ or NO₃⁻, the microbial communities were more diverse and did not select exclusively for full denitrifiers. N₂O alone appears to select for a “generalist” community with full denitrification pathways and lower diversity. In terms of denitrification genes, the combination of acceptors with N₂O appeared to increase the number of microbes carrying nirK, while fully denitrifying bacteria appear more likely to carry nirS. Lastly, all the taxa in NO₂⁻ and N₂O samples were predicted to harbor nosZ. This suggests the potential for reduced N₂O emissions in denitrifying systems.