Biofilm, complex structures formed by microorganisms within an extracellular polymeric matrix, pose significant challenges in the sector by harboring dangerous pathogens and complicating decontamination, thereby increasing the risk of foodborne illnesses. This article provides a comprehensive review of the sigma factor, rpoS’s role in biofilm development, specifically in gram-negative bacteria, and how the genetic, environmental, and regulatory elements influence rpoS activity with its critical role in bacterial stress responses. Our findings reveal that rpoS is a pivotal regulator of biofilm formation, enhancing bacterial survival in adverse conditions. Key factors affecting rpoS activity include oxidative and osmotic stress and nutrient availability. Understanding rpoS-mediated regulatory pathways is essential for developing targeted biofilm management strategies to improve food quality and safety. Furthermore, a bibliometric analysis highlights significant research trends and gaps in the literature, guiding future research directions. Future research should focus on detailed mechanistic studies of rpoS-mediated biofilm regulation, the development of specific rpoS inhibitors, and innovative approaches like biofilm-resistant surface coatings. This knowledge can lead to more effective contamination prevention and overall food safety enhancements.