Background: A multinational outbreak of Mycobacterium chimaera endocarditis following cardiac surgery has been attributed to the use of heater-cooler units (HCUs) during cardiopulmonary bypass. It is hypothesized that mycobacteria can be transmitted to the surgical site via the aerosolization of contaminated water from within the unit. In the United Kingdom, M. chimaera infections have been linked to 1 specific make and model of HCU, which was shown to generate microbial aerosols when circulating water. The manufacturer has since modified this HCU and claims that the dispersal of aerosols has now been prevented. M. chimaera is a common contaminant of HCUs, regardless of make, model, and manufacturer. To help inform local decision making, hospitals require evidence that this modified HCU and/or alternative heater-cooler systems can reduce the risk of mycobacterial infection by incorporating design features that prevent the generation of microbial aerosols external to the device. The time required to culture M. chimaera means investigations focusing on naturally or artificially contaminated HCUs are problematic. Instead, specialist aerobiological techniques incorporating a nonpathogenic, aerostable, biological tracer (Bacillus atrophaeus) were used to investigate microbial aerosols generated and released from brand-new and ‘upgraded’ HCUs. Methods: 4 HCUs (A–D), supplied directly by the manufacturers, were filled with filtered tap water, and high numbers of B. atrophaeus (109 CFU/L) were added to the tanks. High-volume cyclone samplers were used to sample the air when each HCU was switched off and during different operational phases. Samplers were operated for 5 minutes and the collecting fluid cultured for B. atrophaeus. The number of colonies was converted to CFU per cubic meter of air. Results: Under controlled experimental conditions, HCU-A released a small but significant level of aerosol during operational phases (eg, cooling) that resulted in increased pressure within the tank. The filler flap was identified as the principal area of aerosol release. The circulation of water within HCU-B and HCU-C was shown to generate an aerosol but, when connected to an ‘aerosol collection set,’ this aerosol was not released. However, it is essential that effective and sufficient vacuum is maintained. There was no aerosol release from HCU-D. Conclusions: A specialist in aerobiology using a biological tracer can determine the level of aerosol released from an HCU and its location. However, transmission of M. chimaera could occur via aerosolization of contaminated water, but it is not the only possible route of infection. The efficacy of recommended decontamination procedures must also be assured.