The aim of the study was to describe the Mycobacterium chimaera contamination in heater-cooler devices after the application of a protocol of cleaning and disinfection in a tertiary hospital. It was an observational study at the La Paz-Cantoblanco-Carlos III University Hospital, Madrid, Spain. Seven heater-cooler devices are used in our hospital: five 3T Sorin (LivaNova) and two Maquet. We followed the manufacturers' instructions for cleaning and disinfection of the different heater-cooler devices. Environmental testing was developed monthly from January 2017 to July 2019. Samples were obtained from both cardioplegia and patient circuits and before and after the disinfection process and were cultured in appropriate media for non-tuberculous mycobacteria and heterotrophic bacteria (coliforms and Pseudomonas aeruginosa). A total of 320 samples were taken. Mycobacterium chimaera grew in four water samples (1.25%) from three different heater-cooler devices, with two positive results occurring after disinfection. The heterotrophic bacteria Delftia acidovorans and Stenotrophomonas maltophilia were also found. There has not been a case of M. chimaera infection in patients after cardiac surgery in our hospital. In March 2019, we decided to move the heater-cooler device outside the operating room. Mycobacterium chimaera contamination is not always eradicated by disinfection processes. We believe that placing 3T heater-cooler devices outside the operating room is the best option in preventing M. chimaera infection during cardiac surgery.