AimsTo determine whether strains of Geobacillus stearothermophilus isolated from a milk powder manufacturing plant were different in their ability to form biofilms and produce spores. In addition, this study evaluated whether there were other physiological characteristics that could differentiate these strains. Methods and ResultsTen G.stearothermophilus strains and one Anoxybacillus species were isolated from a milk powder manufacturing plant. A microtitre plate assay was used to show that these strains differed in their abilities to form biofilms and produce spores. Scanning electron microscopy showed differences in the biofilm morphologies of three of the G.stearothermophilus strains. Biochemical profiling, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and fatty acid profiling further showed that they had distinct characteristics. ConclusionsThese G.stearothermophilus strains, isolated from the same environment, showed differences in their ability to form biofilms and produce endospores. Based on the multiple characterization methods used in this study, these strains of G.stearothermophilus isolated from one manufacturing plant are diverse. Significance and Impact of the StudyDifferences in the ability of G.stearothermophilus to form biofilms and produce spores may influence the cleaning method used to control the growth of thermophilic bacilli in a dairy processing environment.