Free-living amoebae and Acanthamoebae are known to harbour a range of opportunistic microbial pathogens such as Legionellae, sequestering them from antimicrobial agents as well as environmental stresses. Less is known however of the interaction between the thermotolerant free-living amoebae and Legionellae. In the current study, such phenomena were investigated between an environmental and clinical thermotolerant Acanthamoebae isolate and 6 Legionellae; L. anisa, L. birminghamiensis, L. bozemanii, L. dumoffii, L. erythra and L. pneumophila. All Legionellae could be located within either Acanthamoeba isolate, with L. erythra, and L. pneumophila found located within vacuoles. At concentrations exceeding 2 mg/l, free chlorine was a better disinfectant than combined chlorine against Acanthamoebae-bound Legionellae, though thermal treatment was the most effective of the treatment types investigated. While the interaction with free-living Acanthamoebae increased the resistance of Legionellae to thermal treatment, it increased the sensitivity of Legionellae to free and combined chlorine. Interaction with biofilms did not affect the sensitivity of sessile and intracellular Legionellae to disinfection, caused in part by the thin coverage of biofilm on coupon surfaces. Acanthamoebae cysts remained viable after treatment with 100 mg/l chlorine (free and combined) for 10 min, as well as 80 degrees C, implying that conventional hyper-disinfection may be insufficient for long-term control of Acanthamoebae-bound Legionellae in water distribution systems.