Free-living amoebae might be pathogenic by themselves and be a reservoir for bacterial pathogens, such as Legionella pneumophila. Not only could amoebae protect intra-cellular Legionella but Legionella grown within amoebae could undergo physiological modifications and become more resistant and more virulent. Therefore, it is important to study the efficiency of treatments on amoebae and Legionella grown within these amoebae to improve their application and to limit their impact on the environment. With this aim, we compared various water disinfectants against trophozoites of three Acanthamoeba strains and L. pneumophila alone or in co-culture. Three oxidizing disinfectants (chlorine, monochloramine, and chorine dioxide) were assessed. All the samples were treated with disinfectants for 1 h and the disinfectant concentration was followed to calculate disinfectant exposure (Ct). We noticed that there were significant differences of susceptibility among the Acanthamoeba strains. However no difference was observed between infected and non-infected amoebae. Also, the comparison between the three disinfectants indicates that monochloramine was efficient at the same level towards free or co-cultured L. pneumophila while chlorine and chlorine dioxide were less efficient on co-cultured L. pneumophila. It suggests that these disinfectants should have different modes of action. Finally, our results provide for the first time disinfectant exposure values for Acanthamoeba treatments that might be used as references for disinfection of water systems.