Though the biofilms of thermophilic, endospore-forming bacilli have received increasing interest in recent years, their biofilms are still unclear. In the current study, a comparative characterization of biofilm-forming patterns of totally 105 isolates and reference strains belonging to Anoxybacillus (85) and Geobacillus (20) genera were investigated. Various species belonging to these two genera have been screened for their biofilm production responses in milk, and their biofilm production patterns were also compared with their lactose and casein utilization. Thereby, this is the first report which displays strong biofilm producing behaviours of some thermophilic species in milk such as G. thermoglucosidans, A. caldiproteolyticus, A. suryakundensis, A. salavatliensis, and A. kamchatkensis subsp. asaccharedens except for well-known members like G. steraothermophilis and A. flavithermus. It was also assigned that elevated incubation temperature (65°C) had a stimulative effect on both biofilms forming capacities of Geobacillus and Anoxybacillus. Nevertheless, no direct correlation was found between biofilm formation capacity and lactose or casein catabolism characteristics. Finally, crystal violet binding assay, allowing the determination of the biofilm production amounts of microorganisms relatively in a short time, has been optimized. In this context, a methodological contribution has been provided to the literature in order to determine the possible biofilm production responses of thermophilic biofilms in the dairy industry.