This review discusses the role of extracellular polymeric substances (EPS), primarily polysaccharides, in bacterial biofilm formation. EPS are complex mixtures of biopolymers that form the matrix of biofilms, enabling microbial attachment to surfaces and protection against environmental stress. The review highlights the importance of EPS in various fields, including biomedical, biotechnology, and industrial applications. It discusses the structural variations of EPS produced by different bacterial species and their biotechnological applications. The review also explores the regulatory mechanisms, such as quorum sensing, that control EPS production and biofilm formation. Additionally, it covers the role of EPS in bioremediation and bioleaching, with a focus on Acidithiobacillus ferrooxidans. The review also discusses the chemical composition of EPS produced by A. ferrooxidans and their implications in biotechnology. Finally, it outlines novel techniques for studying biofilm-specific polysaccharides, such as advanced microscopy and spectroscopy methods. The review concludes that understanding EPS and biofilm formation is crucial for improving bioleaching efficiency and developing new biotechnological applications.This review discusses the role of extracellular polymeric substances (EPS), primarily polysaccharides, in bacterial biofilm formation. EPS are complex mixtures of biopolymers that form the matrix of biofilms, enabling microbial attachment to surfaces and protection against environmental stress. The review highlights the importance of EPS in various fields, including biomedical, biotechnology, and industrial applications. It discusses the structural variations of EPS produced by different bacterial species and their biotechnological applications. The review also explores the regulatory mechanisms, such as quorum sensing, that control EPS production and biofilm formation. Additionally, it covers the role of EPS in bioremediation and bioleaching, with a focus on Acidithiobacillus ferrooxidans. The review also discusses the chemical composition of EPS produced by A. ferrooxidans and their implications in biotechnology. Finally, it outlines novel techniques for studying biofilm-specific polysaccharides, such as advanced microscopy and spectroscopy methods. The review concludes that understanding EPS and biofilm formation is crucial for improving bioleaching efficiency and developing new biotechnological applications.