The article reviews the role of extracellular polymeric substances (EPS) in bacterial biofilm formation and their significance in various biomedical, biotechnology, and industrial applications. EPS, primarily composed of polysaccharides, proteins, nucleic acids, lipids, and humic substances, form the matrix of microbial aggregates, facilitating initial cell attachment and protection against environmental stress. The review highlights the regulatory mechanisms, such as quorum sensing (QS), that control EPS production and biofilm development. It also discusses the diverse structural variations of EPS produced by different bacterial lineages and their applications in bioremediation, bioleaching, and other fields. Additionally, the article explores novel techniques for studying biofilm-specific polysaccharides, including atomic force microscopy, confocal laser scanning microscopy, and optical coherence tomography, which provide insights into the complex interactions within biofilms. The conclusion emphasizes the need for further research to enhance our understanding of biofilm systems and their potential in enhancing bioleaching efficiency.The article reviews the role of extracellular polymeric substances (EPS) in bacterial biofilm formation and their significance in various biomedical, biotechnology, and industrial applications. EPS, primarily composed of polysaccharides, proteins, nucleic acids, lipids, and humic substances, form the matrix of microbial aggregates, facilitating initial cell attachment and protection against environmental stress. The review highlights the regulatory mechanisms, such as quorum sensing (QS), that control EPS production and biofilm development. It also discusses the diverse structural variations of EPS produced by different bacterial lineages and their applications in bioremediation, bioleaching, and other fields. Additionally, the article explores novel techniques for studying biofilm-specific polysaccharides, including atomic force microscopy, confocal laser scanning microscopy, and optical coherence tomography, which provide insights into the complex interactions within biofilms. The conclusion emphasizes the need for further research to enhance our understanding of biofilm systems and their potential in enhancing bioleaching efficiency.