Water-borne opportunistic pathogens are responsible for a range of infections in immunocompromised and vulnerable patients, especially in healthcare facilities. Since 2013, over 100 cases of Mycobacterium chimaera prosthetic valve endocarditis and disseminated disease were notified in Europe and the USA, linked to contaminated heater–cooler units used during cardiac surgery. There is the potential for non-tuberculous mycobacteria bacteria to grow in the water tank in the heater–cooler unit. The purpose of this study is to clinically prove that ECMOLIFE HC heater–cooler ordinary disinfection procedure is effective in the prevention of Mycobacterium Chimaera contamination. ECMOLIFE HC heater–cooler was used on seven Extracorporeal Membrane Oxygenation (ECMO) procedures with mean duration of 154 ± 18 (hours). Seven water samples have been collected pre and post disinfection procedure. As disinfection procedure the ordinary was followed according to ECMOLIFE HC User Manual. Each sample was subjected to laboratory test for research by means of Real-Time PCR system of DNA M. Chimaera strain (extraction and quantification). During the clinical practice on seven ECMO procedures involving the use of ECMOLIFE HC, no Mycobacterium chimaera contamination was found in pre- and post-disinfection samples. The test can be considered pass as during normal clinical practice the ECMOLIFE HC resulted not contaminated by Mycobacterium Chimaera. Molecular analysis of all sampled solutions showed no detection of Mycobacterium Chimaera. According to ECMOLIFE HC User Manual, the disinfection method is proved to effectively disinfect ECMOLIFE HC.