The oxygen-limited autotrophic nitrification/denitrification (OLAND) process comprises one-stage partial nitritation and anammox, catalyzed by aerobic and anoxic ammonium-oxidizing bacteria (AerAOB and AnAOB), respectively. The goal of this study was to investigate whether quorum sensing influences anoxic ammonium oxidation in an OLAND biofilm, with AnAOB colonizing 13% of the biofilm, as determined with fluorescent in situ hybridization (FISH). At high biomass concentrations, the specific anoxic ammonium oxidation rate of the OLAND biofilm significantly increased with a factor of 1.5 +/- 0.2 compared to low biomass concentrations. Supernatant obtained from the biofilm showed no ammonium-oxidizing activity on itself, but its addition to low OLAND biomass concentrations resulted in a significant activity increase of the biomass. In the biofilm supernatant, the presence of long-chain acylhomoserine lactones (AHLs) was shown using the reporter strain Chromobacterium violaceum CV026, and one specific AHL, N-dodecanoyl homoserine lactone (C-12-HSL), was identified via LC-MS/MS. Furthermore, C-12-HSL was detected in an AnAOB-enriched community, but not in an AerAOB-enriched community. Addition of C-12-HSL to low OLAND biomass concentrations resulted in a significantly higher ammonium oxidation rate (p<0.05). To our knowledge, this is the first report demonstrating that AHLs enhance the anoxic ammonium oxidation process. Future work should confirm which species are responsible for the in situ production of C-12-HSL in AnAOB-based applications.