Attached growth biological treatment systems are a promising solution to ammonia removal in cold‐temperature climates. Environmental scanning electron microscopy (ESEM) and confocal laser scanning microscopy in combination with fluorescent in situ hybridization (FISH) was used to investigate the effects of 4 months of exposure to 4 °C on nitrifying biofilm and biomass. These molecular and microscopic methods were modified to minimize loss of mass and distortion of in situ perspectives. Environmental scanning electron microscopy revealed that nitrifying biofilm did not exhibit significant changes in volume with exposure to 4 °C. Confocal laser scanning microscopy in combination with FISH showed that the number of ammonia‐oxidizing bacteria (AOB) cells present in the biofilm was statistically consistent during exposure to 4 °C. The RNA content of AOB cells remained sufficient for FISH enumeration. The number of nitrite‐oxidizing bacteria cells remained steady during exposure to 4 °C; however, the RNA content of the cells appeared to decrease with exposure to 4 °C, thereby preventing their enumeration using FISH.