Aims : Free‐living amoebae (FLA) in aqueous systems are a problem for water network managers and health authorities because some are pathogenic, such as Naegleria fowleri, and they have also been reported to operate as reservoirs and vectors of several pathogenic bacteria. Therefore, to better control the occurrence of such amoebae, we evaluate the efficacy of monochloramine against planktonic forms (trophozoites and cysts) and also biofilm‐associated cells of N. fowleri as FLA are often associated with biofilms. Methods and results : From a freshwater biofilm growing in a pilot reactor and inoculated with N. fowleri, we obtained Ct values ranging from 4 to 17 mg Cl2 min l−1 at 25°C and pH 8·2 on both planktonic and biofilm associated cells. In addition, the inactivation pattern of biofilm associated was intermediate between those of trophozoïtes and cysts. Conclusions : The monochloramine efficiency varies with the life stage of N. fowleri (trophozoïte, cyst, and biofilm‐associated). The sensitivity to disinfectant of amoeba, that is, trophozoïtes and cysts, in the biofilm life stage is as high as that of their planktonic cyst form. Significance and impact of the study : This study gives Ct values for cysts and biofilm‐associated N. fowleri. This may impact on water treatment strategies against amoebae and should be considered when controlling N. fowleri in man‐made water systems such as cooling towers or hot water systems.