Biofilms are a major cause of persistent infections, as they are resistant to antibiotics and host defenses. This review discusses the mechanisms of biofilm tolerance, focusing on the role of the biofilm matrix and the physiology of embedded cells. Biofilms exhibit heterogeneity in metabolic activity, leading to varying susceptibility to antimicrobial agents. Nutritional starvation and high cell density contribute to antimicrobial tolerance in both biofilms and planktonic cultures. Recent studies have shown that stress response genes, quorum sensing, and phase variation play key roles in biofilm tolerance. Biofilm bacteria are protected from antibiotics and immune responses due to the matrix, which limits antibiotic penetration and prevents immune cell engulfment. The biofilm matrix also supports biofilm maturation and protects bacteria from phagocytosis. Biofilm tolerance is also linked to restricted antibiotic diffusion and the expression of efflux pumps. The physiology of biofilm-embedded bacteria is similar to that of stationary-phase planktonic cells, which are affected by nutrient limitation and high cell density. Biofilm tolerance is not a genetic change but a reversible phenotype. Biofilms can be cleared by antibiotics if surface layers are targeted, but persistent bacteria in deeper layers may remain resistant. The survival of some bacteria in biofilms is due to heterogeneity in growth rates and the presence of persister cells. Biofilm tolerance is also influenced by stress response genes, which can protect bacteria from antibiotics and the immune system. Biofilms facilitate the spread of antibiotic resistance through horizontal gene transfer. Understanding the mechanisms of biofilm tolerance is crucial for developing effective treatments for biofilm-related infections. Current research suggests that combined treatments may be necessary for biofilm eradication.Biofilms are a major cause of persistent infections, as they are resistant to antibiotics and host defenses. This review discusses the mechanisms of biofilm tolerance, focusing on the role of the biofilm matrix and the physiology of embedded cells. Biofilms exhibit heterogeneity in metabolic activity, leading to varying susceptibility to antimicrobial agents. Nutritional starvation and high cell density contribute to antimicrobial tolerance in both biofilms and planktonic cultures. Recent studies have shown that stress response genes, quorum sensing, and phase variation play key roles in biofilm tolerance. Biofilm bacteria are protected from antibiotics and immune responses due to the matrix, which limits antibiotic penetration and prevents immune cell engulfment. The biofilm matrix also supports biofilm maturation and protects bacteria from phagocytosis. Biofilm tolerance is also linked to restricted antibiotic diffusion and the expression of efflux pumps. The physiology of biofilm-embedded bacteria is similar to that of stationary-phase planktonic cells, which are affected by nutrient limitation and high cell density. Biofilm tolerance is not a genetic change but a reversible phenotype. Biofilms can be cleared by antibiotics if surface layers are targeted, but persistent bacteria in deeper layers may remain resistant. The survival of some bacteria in biofilms is due to heterogeneity in growth rates and the presence of persister cells. Biofilm tolerance is also influenced by stress response genes, which can protect bacteria from antibiotics and the immune system. Biofilms facilitate the spread of antibiotic resistance through horizontal gene transfer. Understanding the mechanisms of biofilm tolerance is crucial for developing effective treatments for biofilm-related infections. Current research suggests that combined treatments may be necessary for biofilm eradication.