Live probiotic bacteria obtained with food are thought to have beneficial effects on a mammalian host, including their ability to reduce intestinal colonization by pathogens. To ensure the beneficial effects, the probiotic cells must survive processing and storage of food, its passage through the upper gastrointestinal tract (GIT), and subsequent chemical ingestion processes until they reach their target organ. However, there is considerable loss of viability of the probiotic bacteria during the drying process, in the acidic conditions of the stomach, and in the high bile concentration in the small intestine. Bacillus subtilis, a spore-forming probiotic bacterium, can effectively maintain a favorable balance of microflora in the GIT. B. subtilis produces a protective extracellular matrix (ECM), which is shared with other probiotic bacteria; thus, it was suggested that this ECM could potentially protect an entire community of probiotic cells against unfavorable environmental conditions. Consequently, a biofilm-based bio-coating system was developed that would enable a mutual growth of B. subtilis with different lactic acid bacteria (LAB) through increasing the ECM production. Results of the study demonstrate a significant increase in the survivability of the bio-coated LAB cells during the desiccation process and passage through the acidic environment. Thus, it provides evidence about the ability of B. subtilis in rescuing the desiccation-sensitive LAB, for instance, Lactobacillus rhamnosus, from complete eradication. Furthermore, this study demonstrates the antagonistic potential of the mutual probiotic system against pathogenic bacteria such as Staphylococcus aureus. The data show that the cells of B. subtilis possess robust anti-biofilm activity against S. aureus through activating the antimicrobial lipopeptide production pathway.