Biofilms are surface-associated microbial communities surrounded by an extracellular polymeric substance (EPS) matrix, and are a key factor in chronic infections. Pathogens such as Pseudomonas aeruginosa, Haemophilus influenzae, Streptococcus pneumoniae, Staphylococcus aureus, and enteropathogenic Escherichia coli are associated with biofilm formation. Biofilms allow bacteria to survive in hostile environments and persist in the host, often resisting antibiotic treatment and host immune responses. Biofilm infections are particularly challenging to diagnose because traditional culture methods are not reliable for detecting biofilm-associated bacteria. The 'Parsek–Singh criteria' have been proposed to help identify biofilm infections in clinical specimens, including surface-associated microbial aggregates and resistance to antibiotic treatment despite the susceptibility of planktonic bacteria. Biofilms are found in various chronic infections, including chronic otitis media, cystic fibrosis pneumonia, chronic rhinosinusitis, and chronic wound infections. Biofilm formation is a complex process involving coordinated bacterial behavior, and biofilms can exist in various forms, including intracellular and extracellular. Biofilm infections are difficult to treat due to their resistance to antibiotics and host defenses, and understanding their role in disease is crucial for developing effective treatment strategies. The study of biofilms in clinical specimens has provided insights into their role in chronic infections and the challenges of diagnosing and treating biofilm-related diseases.Biofilms are surface-associated microbial communities surrounded by an extracellular polymeric substance (EPS) matrix, and are a key factor in chronic infections. Pathogens such as Pseudomonas aeruginosa, Haemophilus influenzae, Streptococcus pneumoniae, Staphylococcus aureus, and enteropathogenic Escherichia coli are associated with biofilm formation. Biofilms allow bacteria to survive in hostile environments and persist in the host, often resisting antibiotic treatment and host immune responses. Biofilm infections are particularly challenging to diagnose because traditional culture methods are not reliable for detecting biofilm-associated bacteria. The 'Parsek–Singh criteria' have been proposed to help identify biofilm infections in clinical specimens, including surface-associated microbial aggregates and resistance to antibiotic treatment despite the susceptibility of planktonic bacteria. Biofilms are found in various chronic infections, including chronic otitis media, cystic fibrosis pneumonia, chronic rhinosinusitis, and chronic wound infections. Biofilm formation is a complex process involving coordinated bacterial behavior, and biofilms can exist in various forms, including intracellular and extracellular. Biofilm infections are difficult to treat due to their resistance to antibiotics and host defenses, and understanding their role in disease is crucial for developing effective treatment strategies. The study of biofilms in clinical specimens has provided insights into their role in chronic infections and the challenges of diagnosing and treating biofilm-related diseases.