Abstract: To investigate the effect of free nitrous acid (FNA) inhibition on the activity of high-efficiency nitrifying bacteria (ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB)), a continuous-flow reactor was conducted at ammonia concentration gradients (50 mg/L→80 mg/L→100 mg/L→150 mg/L→200 mg/L→250 mg/L→300 mg/L→350 mg/L→400 mg/L→450 mg/L), resulting in a high nitrification rate of 89 mg/(L·h). High-throughput analysis showed that Nitrosomonas (31.02%) increased by 72.13 times compared with the raw sludge. At the same time, Nitrospira (4.36%) increased by 2.03 times. The effects of different FNA concentrations on AOB and NOB activity were investigated by using the sludge. Subsequently, the effective FNA concentration obtained was applied for sludge with different nitrification rates. Results show that FNA inhibition can dramatically inhibit AOB and NOB activity. When the FNA concentration maintains at 0.5 or 0.6 mg/L, high activity happens in AOB and NOB. The NOB activity is gradually reduced to 0% and AOB activity still maintains above 56% at an FNA concentration of 0.7 mg/L. The AOB activity is severely inhibited at an FNA level of 0.9 mg/L. Therefore, the FNA concentration of 0.7 mg/L can significantly inhibit NOB activity when the nitrification rates are between 15 and 89 mg/(L·h). However, FNA values should be increased to inhibit NOB activity and retain AOB activity as the nitrification rate increases.