Aim: To prevent further outbreaks of hypersensitivity pneumonitis (HP), biocides are required which are capable of protecting water-based coolants from proliferating mycobacteria. The aim of this study was therefore, to test different biocide preparations on their mycobactericidal activity. Methods and results: Minimal inhibitory concentration values were determined for Mycobacterium chelonae and Mycobacterium immunogenum for triazine-based, methyloxazolidine-based, N/O-formal-based biocidal formulations. All biocides were effective already at a low dosage (< 0.05%) irrespective of the presence or absence of organic soiling, except for one N/O-formal-based formulation containing Kathon 886 (CMI). Quenching of CMI in the presence of organic soiling was found to account for loss in efficacy as determined by high-performance liquid chromatography measurement. Preservation tests were carried out to investigate the efficacy of the biocidal preparations under practical conditions. Conclusions: Results indicate that methyloxazolidine-based biocidal preparations were most effective to prevent coolants from microbial contamination including rapidly growing mycobacteria. Furthermore, it could be demonstrated that common dipslides can be used to easily monitor coolants for contamination by mycobacteria. Significance and Impact of the Study: Our data does not support the hypothesis that mycobacterial proliferation is enhanced by the reduction of competitive microbial population by biocides such as triazines as decribed earlier but rather suggests a protective effect of biocides regarding mycobacteria in the presence of competitive microbial flora, thereby preventing further outbreaks of HP.