Abstract Aims Thermophilic spoilage bacteria and their biofilms formed during milk powder processing posed threats to safety and quality of dairy products. This research aims to understand more about the bacterial behaviours and their social models in biofilms. Methods and Results Interactional effects from both extracellular metabolites and co-culture on biofilms formation of the contaminating thermophilic bacteria were determined. The results showed that strong biofilm formers always had high AI-2 activities, including Geobacillus stearothermophilus gs1, Bacillus licheniformis bl1 and Thermoactinomyces vulgaris tv1. Metabolites from themself or other species altered their biofilm biomass detected by crystal violet staining. Dual-species cultures observed by confocal laser scanning microscope indicated either synergistic or inhibitory effects between B. circulans bc1 and G. stearothermophilus gs1, as well as B. licheniformis bl1 and G. stearothermophilus gs1. Fourier transform infrared spectrometry results revealed the significant diversities in polysaccharides of the biofilm matrix. Conclusions Cell communication played an important role on biofilm formation in the complex microbial community. Intraspecific and interspecific extracellular metabolites influenced collective bacterial behaviours under mixed circumstances. Significance and impact of study This research provided evidences on cell communication and biofilm formation of thermophilic bacteria in dairy industry.