AimsTo determine the minimum inhibitory concentrations (MIC) of a range of cleanroom fungi against three disinfectants common to the pharmaceutical and healthcare sectors: biguanide (chlorhexidine) and two quaternary ammonium compounds (benzalkonium chloride and cetrimide). Methods and ResultsThe in vitro fungicidal activities of the three biocides were studied against 112 cleanroom fungal isolates using broth microdilution technique (CLSI M38-A2 standard). ConclusionsMinimum inhibitory concentration (MIC) for all three biocides against hyaline fungi showed results of not more than 16gml(-1). Alternaria showed <32gml(-1) and other dematiaceous fungi reported that 8-16gml(-1) for biguanides and QACs. This study clearly demonstrates that the most frequently isolated micro-organisms from an environmental monitoring programme may be periodically subjected to broth microdilution testing with cleanroom disinfectant agents used in the disinfection programme confirm their sensitivity profile. Significance and Impact of the StudyNo large collection of data exists on the activity of biocides on pharmaceutical cleanroom fungal isolates. This is the first study report with large collection of cleanroom fungal isolates tested against common biocides using the broth microdilution antifungal susceptibility testing to determine the MIC value. The data presented support a quality control procedure for cleanroom disinfection.