Microbial biofilm is defined as a group of microorganisms adhered to an animate or inanimate surface through a self-produced exocytosed polymeric substance. The inability of the antimicrobial components to infiltrate the composite biofilm grid or to the make-up of the bacterial arrangement inside the biofilm helps in protecting the microorganisms. Biofilms can protect microorganisms from extremities: high temperature, pH, high saltiness, high pressure, scarcity of nutrients, antibiotics, etc. as a “protective covering.” In an ecological biome, establishment of biofilms has a crucial role in microbial persistence. Biofilms create challenges in manufacturing sectors like water, energy, food, and medicine, as they build up on the food itself and food-processing equipment, contaminating water channels, corroding submerged metal surfaces, and persisting on medical use devices. Biofilm can boost resistance to host protection systems and are projected for implicating persistent infections, surging healthcare expenses, inpatient care rates, sickness, and deaths. Biofilm-related ailments consist of chronic otitis, periodontitis, respiratory diseases, and chronic wound infections. Bacterial cells inhabiting in the interior of a biofilm are resilient to antibiotics, enduring up to 100–1000 fold better than individual cells. The microbiota inhabiting the human gut creates an intricated-interdependent collaboration with epithelial and immune cells of the gastrointestinal tract (GIT). During the course of collaboration, the microbiota provides nourishment, establishes an initial line of security countering invasion by pathogenic organisms, modulating epithelial growth and immune retaliation. When bacterial cells shift to the biofilm mode of growth, it undergoes a phenotypic modification in behavior in which hefty number of genes are differentially regulated. In this chapter, the regulation of certain genes leading to formation of biofilms by different microorganisms is discussed.