The importance of non‐tuberculosis mycobacterial biofilm species in medicine, industry and the environment has recently gained attention. Our objectives were to characterize biofilm growth of Mycobacterium phlei M4, as a model of rapidly growing mycobacteria using the minimal biofilm eradication concentration (MBEC™) and to compare biocide susceptibility of planktonic and biofilm organisms. Scanning electron microscopy was also carried out to observe biofilm morphology. With the exception of Sporicidin? and Virkon? the minimum bactericidal concentration values for all biocides tested were lower than the MBEC values. The MBEC™ assay system was seen to produce multiple and reproducible biofilms of M. phlei and to be a useful tool for susceptibility studies.