Inactivation and adaptation of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) to free nitrous acid (FNA) was investigated. Batch test results showed that AOB and NOB were inactivated when treated with FNA. After an 85-day operating period, AOB in a continuous pre-denitrification reactor did not adapt to the FNA that was applied to treat some of the return activated sludge. In contrast, NOB did adapt to FNA. NOB activity in the seed sludge was only 11% of the original activity after FNA batch treatment, at 0.75 mg HNO2-N/L. NOB activity in the pre-denitrification reactor was not affected after being exposed to this FNA level. Nitrosomonas was the dominant AOB before and after long-term FNA treatment. However, dominant NOB changed from Nitrospira to Candidatus Nitrotoga, a novel NOB genus, after long-term FNA treatment. This adaptation of NOB to FNA may be due to the shift in NOB population makeup.