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Biological nutrient removal from wastewater to reach acceptable levels is needed to protect water resources and avoid eutrophication. The start-up of an anaerobic ammonium oxidation (anammox) process from scratch was investigated in a 20 L sequence batch reactor (SBR) inoculated with a mixture of aerobic and anaerobic sludge at 30 ± 0.5 °C with a hydraulic retention time (HRT) of 2–3 days. The use of NH4Cl, NaNO2, and reject water as nitrogen sources created different salinity periods, in which the anammox process performance was assessed: low (<0.2 g of Cl−/L), high (18.2 g of Cl−/L), or optimum salinity (0.5–2 g of Cl−/L). Reject water feeding gave the optimum salinity, with an average nitrogen removal efficiency of 80%, and a TNRR of 0.08 kg N/m3/d being achieved after 193 days. The main aim was to show the effect of a hydrazine addition on the specific anammox activity (SAA) and denitrification activity in the start-up process to boost the autotrophic nitrogen removal from scratch. The effect of the anammox intermediate hydrazine addition was tested to assess its concentration effect (range of 2–12.5 mg of N2H4/L) on diminishing denitrifier activity and accelerating anammox activity at the same time. Heterotrophic denitrifiers’ activity was diminished by all hydrazine additions compared to the control; 5 mg of N2H4/L added enhanced SAA compared to the control, achieving an SAA of 0.72 (±0.01) mg N/g MLSS/h, while the test with 7.5 mg of N2H4/L reached the highest overall SAA of 0.98 (±0.09) mg N g/MLSS/h. The addition of trace amounts of hydrazine for 6 h was also able to enhance SAA after inhibition by organic carbon source sodium acetate addition at a high C/N ratio of 10/1. The start-up of anammox bacteria from the aerobic–anaerobic suspended biomass was successful, with hydrazine significantly accelerating anammox activity and decreasing denitrifier activity, making the method applicable for side-stream as well as mainstream treatment.