This　work　investigated　the　impact　of　hydrazine　(N2H4),　a　key　intermediate　product　of　the　anaerobic　ammonium　oxidation　(anammox)　process　and　a　proposed　additive　to　enhance　the　metabolism　of　anammox　bacteria,　on　the　hydroxylamine　(NH2OH)　oxidation　process　mediated　by　hydroxylamine　oxidoreductase　(HAO)　in　ammonium-oxidizing　bacteria　(AOB).　Through　batch　tests　using　highly　enriched　AOB-dominated　sludge,　we　demonstrated　that　N2H4　competitively　inhibited　NH2OH　oxidation　while　significantly　stimulating　nitrous　oxide　(N2O)　production.　Molecular　docking　confirmed　the　competitive　inhibition　relationship　between　NH2OH　and　N2H4　for　the　same　active　sites　of　HAO,　verifying　the　higher　binding　affinity　of　NH2OH　(binding　energy:　−2.3　kcal/mol)　compared　with　N2H4　(binding　energy:　−1.7　kcal/mol).　The　interference　of　N2H4　was　further　substantiated　with　dedicated　batch　tests　initiated　with　NH4+.　The　competitive　inhibition　of　≤　10　mg-N/L　N2H4　was　found　to　induce　significant　NH2OH　accumulation,　elevate　N2O　production　and　impair　partial　nitrification　efficiency.　This　work　raised　not　only　awareness　about　the　indirect　negative　impact　of　the　anammox　process　on　the　activity　and　functionality　of　AOB　but　also　concerns　regarding　the　application　of　N2H4　to　enhance　the　activity　of　anammox　bacteria,　particularly　in　partial　nitrification/anammox　systems.　©　2025　Elsevier　B.V.