Background: In 2013, a global outbreak of Mycobacterium chimaera infections due to contaminated heater-cooler units emerged. This ongoing problem has highlighted the question of whether disinfection recommendations for medical devices containing water circuits are adequate for preventing contamination and possible recontamination by nontuberculous mycobacteria. The formation of biofilms in such devices exacerbates the problem. This study aimed to assess the efficacy of disinfectants on biofilms and suspensions of the M. chimaera strain ZUERICH-1, and to compare it with two unrelated M. chimaera strains obtained from different sources. Methods: Disinfection efficacy testing for biofilm was performed using a Bead Assay for Biofilms and for bacteria in suspension according to the European Standard EN 14348. Three different disinfectants, glutaraldehyde, sodium hypochlorite and peracetic acid, were assessed. M. chimaera ZUERICH-1, two genetically unrelated M. chimaera isolates and M. avium subsp. avium ATCC 15769, which is included in European standards for disinfectant testing on mycobacteria, were analyzed. The biofilms’ structure and composition were analyzed by chemical and molecular techniques and advanced imaging methods. Results: We found that peracetic acid and glutaraldehyde in standard concentrations were able to effectively inactivate (≥ 4 log₁₀ reduction) suspended bacteria of all three strains, but chlorine failed in all cases. Formation of biofilm generally enhanced the tolerance of M. chimaera to disinfectants. Peracetic acid in standard concentration could not effectively inactivate biofilms of M. chimaera ZUERICH-1, but was effective against biofilms of the other M. chimaera strains tested. Similarly, glutaraldehyde in standard concentration could not inactivate biofilm of ZUERICH-1. Biomass analysis showed higher amounts of extracellular matrix of ZUERICH-1 when compared to the other two strains. Conclusions: The data suggest that current standard disinfection recommendations do not ensure sustained inhibition of M. chimaera when embedded in biofilm. Additional measures are needed to prevent nosocomial transmission of M. chimaera through contaminated heater-cooler units.