Nitrate is one of the chemicals often added to wastewater to control hydrogen sulfide production by sulfate-reducing bacteria (SRB). While the effect of nitrate in various SRB pure cultures is well documented, the effect observed in mixed microbial communities is not consistent. This study investigates the response of mixed SRB communities to nitrate, by examining the changes in activity and community composition of sulfidogenic wastewater biofilm over a 10-day period with 10 mmol L(-1) nitrate exposure. Biofilms were enriched in SRB belonging to the Desulfobacter, Desulfobulbus, Desulfomicrobium, and Desulfovibrio genera. Nitrate exposure decreased dsrB transcription within 4 h, and sulfate consumption within 10 days, but it did not fully eliminate sulfide production in the biofilms. The effect of nitrate on SRB was genus specific; Desulfobacter and Desulfobulbus disappeared while Desulfovibrio and Desulfomicrobium persisted in the biofilms. Nitrate exposure also led to the rapid proliferation of nitrate-reducing bacteria within the biofilms, and increased the biofilm thickness. Nitrate consumption began within 2 h of nitrate exposure and gradually increased in rate over time. Transcription of the nitrate reductase napA, and the diversity of nitrate reductase genes narG and napA also increased concurrently. Our results demonstrate that some SRB, presumably those able to tolerate or detoxify nitrite, will persist in sulfidogenic wastewater biofilms despite continuous exposure to high levels of nitrate. Nitrate is therefore unlikely to provide lasting hydrogen sulfide suppression in wastewater biofilms harboring Desulfovibrio or Desulfomicrobium populations.