Monochloramine penetrated fully into nitrifying biofilms within 24 hours when fed at a 4:1 Cl2:N ratio, showing a cessation of aerobic activity via DO penetration following application of monochloramine. However, monochloramine penetration did not necessarily equate to a loss in viability, and the presence of excess ammonia in the water system prevented microbial inactivation. Biofilm recovery occurred when disinfection stopped. Monochloramine showed greater penetration compared to chlorine. Monochloramine penetrated into the biofilm surface layer 49 times faster than chlorine within the nitrifying biofilm and 39 times faster in the multi-species biofilm than did chlorine.